In both the original comics and the live-action films, Splinter is a pet rat of ninja Hamato Yoshi in Japan, and uniquely intelligent for his species. Yoshi becomes embroiled in a deadly dispute with fellow ninja Oroku Nagi over a woman named Tang Shen and is eventually murdered in turn by Nagi's brother, Oroku Saki, as revenge for his brother's death. However, in the 1990 film and 2003 TV series, Nagi is removed entirely. Additionally, in the 1990 film, Splinter escapes from his cage during the murder, and attacks Saki, clawing at his face. Saki, in turn, slices his ear off. It is suggested that this mutilation is the reason Saki took the Shredder disguise to hide the scars. In contrast, in the 2003 animated series, it was Hun's face which Splinter clawed.
Without a home, Splinter is forced to run away and live in New York City's sewers. Due to a traffic accident, four baby turtles and a canister of mutagenic ooze are sent down into the sewer. The canister mutates both Splinter and the turtles into humanoid forms; it also enhances the former's already sophisticated intellect. Splinter names the four turtles Leonardo, Donatello, Raphael, and Michelangelo (after a book on Renaissance artists that he found in a storm drain) and trains them in the arts of ninjutsu so that they can avenge his dead master, while raising them as his own sons, as well as educating them in scholastic.
In the 2003 animated series, Splinter possesses a significant reputation as a ninja master; in a multi-part episode, he is revealed to be a champion of the Battle Nexus, a multi-dimension-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 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. Splinter's backstory is told in the episode "Tale of Master Yoshi", which reveals he was a hungry rat that snuck into the home of the Ancient One, the master and adoptive father of Yoshi. He was saved by Tang Shen, Yoshi's love interest and the Ancient One's adopted daughter, who kept him as a pet. After Yoshi killed his former friend Yukio Mashimi for murdering Shen, he moved to New York City with the rat, whom he named Splinter as a reminder of his loss and vengeance.
In the episode "Tempus Fugit", Splinter and the Turtles, who time-traveled into the future, are sent back to the present time, but the villain, Viral, blasts Splinter with a decompiler ray that scatters his bits all over the internet. While Splinter was not seen much in the "Back to the Sewers" season, the focus of those episodes is the Turtles' efforts to find Splinter's data bits by traveling all over cyberspace. Splinter is restored in the final episode and helps defeat the Cyber Shredder.