One year after their battle with Shredder, the Teenage Mutant Ninja Turtles, Leonardo, Donatello, Raphael, and Michelangelo, still live beneath the sewers, having allowed Vern Fenwick to take the credit for Shredder’s defeat. At Grand Central Station, April O’Neil discovers and informs the turtles that scientist Baxter Stockman is working for Shredder and plans to bust him out of prison. As Shredder is transferred between prisons with two criminals alongside him, Bebop and Rocksteady, by corrections officer Casey Jones, the Foot Clan attack the convoy transporting them. Despite the turtles interference, Shredder escapes when Stockman uses a teleportation device. Shredder is hijacked mid-teleport, winds up in another dimension, and meets the alien warlord Krang, who tells him about his plans to invade Earth. He gives Shredder a purple mutagenic compound in exchange for his promise to find three components of a machine that Krang sent to Earth long ago which will open a portal to his dimension when united, knowing that Shredder and Stockman have the first piece. Casey tells the NYPD chief what happened to Shredder but is met with disbelief, and decides to go out on his own.
Shredder returns to New York City, recruits Bebop and Rocksteady, who also escaped, and has Stockman use Krang’s purple mutagen to transform them into powerful animal mutants—a humanoid warthog and rhinoceros. April witnesses their transformation and is able to steal the mutagen vial. Pursued by the Foot, she is rescued by Casey, who uses hockey gear. In the ensuing battle, the vial is taken into police custody. April then introduces Casey to the turtles, and Raphael and Michelangelo make fun of and pull pranks on him. In the lair, Donatello deduces that the mutagen could be used to turn the turtles into humans, enabling them to live normal lives above ground, but Leonardo refuses and insists on keeping it a secret from the others. However, Michelangelo overhears their conversation and tells Raphael, which enrages Raphael and leads to a fierce argument between the brothers. Leonardo benches Raphael and takes Michelangelo off the mission. In the Natural History Museum, Shredder, Bebop, and Rocksteady are able to find the second piece and steal it before Leonardo and Donatello arrive. Still furious, Raphael recruits Michelangelo, April, Casey, and Vern to break into the NYPD police headquarters and retrieve the mutagen. Vern distracts the police while April and Casey retrieve the mutagen, but the Foot arrive ahead of them. In the ensuing battle, the turtles existence is revealed to the police, who react with fear and hatred. April and Casey intervene and help the brothers escape, but are arrested in the process. The police also see on TCRI’s cameras that April stole the mutagen. However, Stockman had edited the tape so that only April is seen.
With Shredder’s forces having already recovered two pieces of the device, the turtles track Bebop and Rocksteady as they recover the final piece in the rainforests of Manaus, Brazil. The turtles board Rocksteady and Bebop’s jet in midair. In ensuing the battle, the jet is critically damaged after Rocksteady fires a tank mounted machine gun within the jet’s cargo hold and crashes into a river. After the Turtles battle with Bebop in the river for control of the piece, Rocksteady emerges in the tank and helps Bebop escape with the piece. The turtles return to New York as Shredder and Stockman complete the portal device and open the gateway to Krang’s dimension through which his modular war machine, the Technodrome, begins to emerge. Shredder betrays Stockman and his men take him away to their headquarters in Tokyo. When entering the Technodrome, Krang likewise betrays Shredder, freezing him and locking him away with his collection of other defeated foes.