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The fugitive Gecko brothers Seth (George Clooney) and Richie (Quentin Tarantino) are fleeing the F.B.I. and Texas police after Richie has broken Seth out of custody at court, in a bloody shootout. Seth Gecko is a cold, tough anti-hero, while his younger brother Richie is a delusional, homicidal serial rapist.

Seth and Richie hold two girls hostage in a liquor store to attempt to get Pete Bottoms, the clerk, to get rid of Texas Ranger Earl McGraw, who has entered the store. Just as McGraw is about to leave, he is shot in the head by Richie, thinking that Pete has mouthed "help us." This leads to an argument between Seth and Richie, and Richie shoots Pete in the shoulder. Enraged, Pete manages to pull out a gun he had hidden in a safe. Though he manages to shoot Richie in the hand, Pete fails to reload quickly enough, and is set on fire by the two brothers (during which the hostages escape). As the two siblings argue and depart, the liquor store quickly catches fire and explodes behind the two, who don't bother to notice. It is revealed during the brothers' argument that Richie was merely supposed to buy a road map from the store.

The Geckos arrive at a motel, along with Gloria Hill, a hostage they had taken from the court in the trunk of their car. Seth warns Hill not to talk or try to escape. Seth then leaves to get food and to scope Mexico's border, while Richie watches a news report about him and his brother.

At the same time, the Fuller family: Jacob (Harvey Keitel), the father and a former pastor who has experienced a crisis of faith, and his two children Scott (Ernest Liu) and Kate (Juliette Lewis) are on a vacation in their RV. Meanwhile, Seth returns with food for the three of them and discovers that Richie has raped and killed Hill. Seeing Hill's nude and battered body lying on the hotel bed, Seth becomes enraged, only to calm down and promise his brother that they will get to Mexico tomorrow.

The Fullers arrive at the motel, only to be kidnapped by the Geckos, who force the Fullers to smuggle the brothers past the Mexican Border. Richie's eyes begin to roam on Kate which makes Seth and Jacob uncomfortable. But both men make an uneasy truce if it means Jacob and his family will come out of the ordeal unharmed and the Geckos can make it past the border. They manage to fool the border agent (Cheech Marin) and arrive at the "Titty Twister", a bar in the middle of a desolate wasteland, where the Geckos have agreed to be picked up by Carlos (Cheech Marin again), their contact, at dawn.

At the "Titty Twister", the Geckos run into a few conflicts. First they encounter Chet Pussy, an obnoxious bouncer (Cheech Marin again) whom Seth and Richie pummel to gain entrance. Then Razor Charlie (Danny Trejo), the owner and main bartender, refuses to serve them, stating that he only serves bikers and truck drivers. Though Jacob manages to prove that he is a truck driver, stating that a truck license is required to drive an RV, Seth nevertheless gets angry when Big Emillio, a bouncer, puts his hand on him. Jacob defuses the situation by buying everyone a drink.

Exotic dancer Santanico Pandemonium (Salma Hayek), the main attraction at the "Titty Twister", appears. After her show is over, Chet the bouncer, having recovered from his beating, enters the bar with Razor Charlie and Big Emilio at his side. Once they attack the Geckos and stabbing Richie in his wounded hand, he and Seth shoot all three to death. Upon seeing Richie's blood soaked hand, Santanico morphs into a horrible creature, jumps on Richie's back and sinks her fangs into his neck, killing him. It soon becomes evident that everyone that works at the "Titty Twister" are vampires.

In the ensuing chaos, nearly every biker and trucker in the bar is killed in a bloodbath. Only, Seth, Jacob, Kate, and Scott survive, along with a biker named Sex Machine (Tom Savini) who has a gun holstered to his crotch, and Frost (Fred Williamson), a Vietnam War veteran who likes cigars. They manage to kill all of the vampires in the room (including Santanico Pandemonium, Razor Charlie, Big Emillio, Chet Pussy, and a vampire called "Mouth Bitch"), but their problems are not yet over. A host of vampires attempt to enter the bar, which is then barricaded. Also, the slain clients - including Richie - are starting to rise again as vampires and must be destroyed. During the struggle, one of the vampires bites Sex Machine in the arm, but he tries to cover up his wound.

The survivors try to decide what to do next. Some of them suggest staying until dawn. When Kate says in ever-growing panic that she cannot stand all of this, Frost disagrees, recalling a traumatic event that occurred to him in Vietnam. While he is telling this story, Sex Machine begins to develop vampire traits and when Frost finishes his story, Sex Machine bites him (non-fatally) on the neck. He also manages to bite Jacob in the arm and throws him against a wall, before being thrown through a door by Frost, causing all of the vampires outside to enter. In the process, Frost also turns into a vampire instantly. Seth, Kate, and Scott quickly escape to a back room.

Meanwhile, Jacob manages to combine a baseball bat and a shotgun in the form of a cross and escapes into the back room where Seth, Scott, and Kate are hiding. They fashion weapons from items found in the storeroom (including carving crosses on the remaining bullets in Seth's gun), and prepare to fight the vampires. Before joining them, Jacob makes his children swear that they will kill him when he inevitably changes into a vampire.

The four then make their assault on the undead. Seth and Kate manage to kill Sex Machine (after he turns into a demon rat), and Jacob attacks Frost. However, just after Frost is vanquished, the other vampires stop attacking Jacob, since Jacob has turned into a vampire himself. He is standing next to Scott, who cannot bring himself to kill Jacob. He bites Scott, who then kills him. Scott is then attacked by several vampires and Kate is forced to shoot him and his attackers. As the vampires have very poor reactions to anything blessed, the bullets blow up their bodies, which blows up Scott as well.

Seth and Kate quickly get surrounded by the vampires, but then streams of sunlight shine through holes in the walls, showing that it is now dawn. Seth and Kate quickly shoot holes into the walls through which sunlight pours in, holding the remaining vampires at bay. Just then, Carlos attempts to enter the building. On Seth's call his bodyguards blast open the door, and the sunlight streaming in in a broad beam (and a disco ball on the ceiling reflecting it into every corner of the bar) blows up all of the remaining vampires.

Seth and Kate flee the building as the vampires go up in flames. Seth berates Carlos for choosing the "Titty Twister" as their rendez-vous and demands that now he will pay Carlos only 15% (rather than 30% as per their prior agreement) because of Richie and the Fullers' deaths. Eventually, the two agree to 25%. Seth leaves with Carlos. Though he is tempted to take Kate with him, he decides he is not evil enough to take her to a town of vicious criminals, where she will surely meet a bad end, stating his famous line, "I may be a bastard, but I'm not a fucking bastard." (This line is parodied by Australian satire band TISM with their song "I Might Be A Cunt, But I'm Not A Fucking Cunt"). He throws some money at her, and allows her to continue her journey without him, though she appears somewhat at a loss to continue her family's vacation after the events of the night.

As they leave, the camera pans back to reveal that the "Titty Twister" was actually the top of a buried ancient Aztec Temple, presumably the home of vampires for centuries, and reveals hundreds of trucks and bikes that have been toppled down the side of the cliff following their owners' untimely demise.