Oscar-nominated actor Mako was announced as the voice of Splinter at San Diego Comic Con the day before he died. Fortunately, he had finished the majority of his acting.
According to writer and director Kevin Munroe on the DVD commentary, the little song that Splinter sings when he enters the kitchen is an old Japanese lullaby that Mako ad-libbed for the scene.
While Leonardo, Raphael, and Donatello all have brown eyes, Michelangelo has blue eyes. According to writer and director Kevin Munroe, this was done to make Michelangelo seem a bit more innocent and younger than the other turtles.
During the fight between the Nightwatcher and the monster in the diner, at one point, a refrigerator falls on top of the monster and the music cuts out and the monster is heard making a whimpering noise. This was actually a mistake in the soundtrack, but when the filmmakers saw it, they couldn't stop laughing, and left it in the movie.