The Turtles slowly come into conflict with the Foot Clan. After their lair is destroyed by Baxter Stockman's mousers, they find a new home elsewhere in the sewers and also gain new allies in the form of April O'Neil and Casey Jones. The Turtles foil the plans of the Shredder, Hun, and Stockman on numerous occasions before Shredder offers Leonardo a peace offering in exchange for their service, citing an unknown evil they must face together. Splinter reveals the Shredder and Hun's hand in murdering his master Hamato Yoshi, and the two sides come to blows in a battle that seemingly ends in the Shredder's defeat. However, unknown to the Turtles, the Shredder survives, and after his ninjas savagely ambush and wound Leonardo, destroys April's home and believes them dead, though the Turtles, Splinter, Casey and April survive and depart for Casey's family's farm house in rural New York. After Leonardo's wounds heal and their resolve is restored, the group returns to New York to face the Foot Clan. However, after their triumph against the Shredder, Splinter vanishes, and after following a lead from a mysterious group known as the "Guardians", the Turtles discover the Utroms, an alien race hiding amongst humanity, and are unable to save Splinter from them before being teleported into space.

The Turtles find themselves teleported to the alien world of D'Hoonibb, where they immediately are caught up in a war between the Triceraton Republic and the Galactic Federation. The Turtles end up on the Triceraton home world, and after saving Honeycutt, are teleported back to Earth by the Utroms. The aliens reveal themselves to be benevolent and reunite the Turtles with Splinter. In the wake of the Shredder's defeat, a gang war erupts over control of his lost territory by the remainder of the Foot Clan, Hun and the Purple Dragons, and the New York mafia, aided by Baxter Stockman. Shredder is revealed to have survived yet again, and Karai's loyalty to her father conflicting with her honor becomes a critical dilemma for her.