As the film opens, Banner works at a soda bottling factory in Rocinha while searching for a cure for his condition (through analyzing the properties of certain rare Amazonian plants and herbs) with the help of a colleague on the Internet, known only as "Mr. Blue", and to whom he is known as "Mr. Green". He is also learning meditative breathing techniques from a martial arts expert, played by Brazilian Jiu-Jitsu legend 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 by transforming into the Hulk and killing every member of the military team, with the exception of Blonsky, inside the bottle factory. After Ross explains how Banner first became the Hulk, a vengeful Blonsky agrees to be injected with a small amount of a new super soldier serum, which gives him enhanced strength, speed, agility, reflexes, endurance and healing.
Meanwhile, Banner returns to Culver University (where the Hulk was born) in Virginia 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, having been tipped off by the suspicious Samson. The Hulk wins the battle, putting Blonsky in the hospital despite his recent "upgrade", and flees with Betty. After he calms down, Banner and Betty go on the run. After several stops, Banner again makes contact with "Mr. Blue", who urges them to travel to New York City to meet him. He turns out to be cellular biologist Dr. Samuel Sterns (Tim Blake Nelson), a college professor. 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 (which he sent from Brazil) into a large supply with the intention of using it to bring humans 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 taken into custody with Betty.
While Sterns is interrogated by a female soldier about his work, Blonsky strikes her down and demands that Sterns inject him with Banner's blood sample, as he covets the awesome power of the Hulk. Sterns warns that the combination of the supersoldier formula (which Blonsky has overdosed on, slightly deforming 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 hideous monster, with strength and power that surpasses that of the Hulk, but retaining Blonsky's intelligence and personality. 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, much to his delight.
Banner, realizing that the Hulk 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. As he falls towards the ground, Banner panics, finally triggering the transformation. Banner's plan succeeds, and after a long and brutal battle through out Harlem, the Hulk defeats Blonsky by nearly strangling him to death with a huge chain, relenting his grip only after Betty's plea. After having a small, peaceful moment with Betty, the Hulk flees. 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 and the change begins, a grin appears on his face, indicating he is starting to master his transformations. Meanwhile, General Ross is drinking his troubles away in a bar when he is approached by industrialist Tony Stark (Robert Downey Jr.), who casually reveals that a "team" is being put together.