Splinter is the master and father figure of the Teenage Mutant Ninja Turtles. In all versions of the story, he is a mutant rat, but his origins often vary depending on the version. In the original comic, Splinter was the pet rat of a ninja named Hamato Yoshi. Exceptionally intelligent for his species, Splinter was able to learn his master's art by mimicking his movements while he practiced.

Yoshi eventually was embroiled in a dispute with a fellow ninja by the name of Oroku Nagi over the love of a woman named Tang Shen. When the woman favored Yoshi, Nagi grew angry and beat her. Yoshi came across them, and slew Nagi in his rage.

The pair escaped to New York City, but they were tracked down and Yoshi was slain by Nagi's younger brother, Saki, who had become The Shredder.

Without a home, Splinter eventually came to live in the city's sewers. One day, he saw a canister of chemicals go flying from a truck. The canister struck a boy in the face after he saved a blind man from being hit by the truck (a reference to the superhero Daredevil), and then struck and shattered a bowl full of four baby turtles carried by a young boy. The now broken canister and turtles fell into the sewers, where Splinter rescued them, also exposing himself to the ooze that had covered the turtles.

In time, the ooze mutated both Splinter and the turtles, increasing their size and intelligence, and making them more humanoid. Eventually, Splinter named the four turtles Leonardo, Donatello, Raphael, and Michelangelo (after his favorite Renaissance artists) and raised them as his own sons, but also trained them in the arts of ninjutsu, so that they may one day avenge the death of Hamato Yoshi and slay the Shredder, who had amassed a powerful organization in the New York underworld.