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A montage during the opening credit sequence details the film's backstory and the origin of the Hulk. General Thaddeus "Thunderbolt" Ross (William Hurt) meets with Dr. Bruce Banner (Edward Norton), the colleague and lover of his daughter Betty (Liv Tyler). He wants him to revive a World War II-era military bio-force project, but tells Banner the goal of the experiment is to make human beings immune to gamma radiation. The experiment fails, transforming Banner into the monstrous Hulk (voiced by Lou Ferrigno), and injuring Betty. Now a fugitive from the United States Army, Banner has been on the run for five years.

As the film opens, Banner works at a soda bottling factory in Brazil while searching for a cure for his condition with the help of a colleague on the Internet, known only as "Mr. Blue". He is also learning meditative breathing techniques from a martial arts expert (Rickson Gracie) to help regulate his pulse rate and keep his anger under control, and has not transformed in 158 days. After Banner cuts his finger, a drop of his blood ends up in one of the bottles, and is eventually ingested by an ill-fated consumer (Stan Lee) in Milwaukee, Wisconsin. This accident points Ross to Banner's location and he sends a team, led by Russian-born British special ops expert Emil Blonsky (Tim Roth), to capture him. Banner escapes Blonsky by transforming into the Hulk and fighting off his team inside the bottle factory. After Ross explains how Banner first became the Hulk, a vengeful Blonsky agrees to be injected with the super soldier serum, giving him enhanced speed, agility, reflexes and healing ability.

Meanwhile, Banner returns to Culver University in the United States and reunites with Betty, who is dating psychiatrist Leonard Samson (Ty Burrell). On the day he decides to leave, Ross and Blonsky's forces attack Banner at Culver University to draw out the Hulk. The Hulk wins the battle and flees with Betty. After he calms down, Banner and Betty travel to New York City to meet "Mr. Blue", who is cellular biologist Dr. Samuel Sterns (Tim Blake Nelson), at a university. They learn that Sterns has developed a possible antidote that may cure Banner's condition, or merely reverse each individual transformation. After a successful test, Sterns reveals that he has synthesized Banner's blood sample into a large supply with the intention of using it to enhance the human condition to the next evolutionary level. Appalled by what Sterns had done and fearful of the Hulk's power falling into the wrong hands, Banner attempts to convince Sterns to destroy the blood supply, but he is attacked by Ross' forces and he and Betty are taken into custody.

As Sterns goes with an interrogation with a female soldier about his work, Blonsky strikes her down and demands Sterns to inject him with Banner's blood sample. Sterns warns that the combination of the supersoldier formula (which Blonsky has overdosed on, mutating his skeleton) and a gamma treatment would be an unpredictable combination that could turn him into an "abomination". Unconcerned, Blonsky forces Sterns to administer the gamma charge, and he mutates into a powerful monster. He knocks Sterns aside and escapes, rampaging through Harlem to draw the Hulk out. At the lab, an irradiated sample of Banner's blood-derivative drips into an open wound on Sterns' temple, causing his cranium to mutate and expand.

Banner, realizing that he is the only one who can stop the monster, convinces General Ross to release him. He falls from Ross' helicopter as it hovers over the city, hoping the fall will trigger a transformation. Banner's plan succeeds, and after a brutal battle, the Hulk defeats Blonsky by nearly strangling him to death with a huge chain, relenting his grip only after Betty's plea. The Hulk then flees the scene with the army in pursuit.

Thirty-one days later, Banner is in Bella Coola, British Columbia. Instead of trying to suppress his transformations, he is attempting to initiate them in a controlled manner. As his eyes turn green, a grin appears on his face. Meanwhile, General Ross is drinking in a bar when he is approached by Tony Stark (Robert Downey Jr.) who reveals that a "team" is being put together.