Without a home, Splinter was forced to run away and live in New York City's sewers. Due to a traffic accident, four baby turtles and a canister of radioactive ooze were sent down into the sewer, where they mutated both Splinter and the turtles. Splinter named the four turtles Leonardo, Donatello, Raphael, and Michelangelo (after a book on Renaissance artists that he found in a storm drain) and trained them in the arts of ninjutsu, while still raising them as his own sons.

In the 2003 TMNT cartoon, he possesses a significant reputation as a ninja master; in a multi-part episode, he was revealed to be a champion of the Battle Nexus, a multi-dimensional-spanning contest where the greatest combatants of various dimensions come together to fight each other for the title of Battle Nexus Champion. When the Turtles first learn of the Battle Nexus, they participate as well, resulting in Splinter forfeiting when called upon to fight Michelangelo, as he wishes to allow his sons the opportunity to fight where he succeeded in the past.