The line appears in Sunday's much-awaited special where Bart Simpson and his family hang around with Stewie and the rest of the "Family Guy" crew, and has already circulated in a trailer for the episode that Fox released online over the summer.
It punctuates a scene in which the incorrigible Bart is instructing Stewie Griffin in the art of the prank phone call. Bart dials the owner of Moe's Tavern and asks whether there is anyone there with the last name Keybum, first name Lee. When Moe calls out to his patrons, asking for a "leaky bum," everyone gets a laugh.
Stewie thinks that's cool, and asks to make his own prank call.
"Hello, Moe?" he says. "Your sister's being raped."
Tim Winter, president of the advocacy group Parents Television Council, said he's a longtime fan of Matt Groening, creator of "The Simpsons," and sought out the trailer when it was released.
"I was blown out of my shoes when I saw the scene with the rape joke in it," Winter said. "It really troubled me."
He said he found it particularly offensive in the context of stories about sexual assaults on college campuses and, most recently, talk about abusive treatment of women by some players in the National Football League. He said when rape is accepted as a punch line for a joke in entertainment, "it becomes less outrageous in real life."
Fox's entertainment division, through a spokeswoman, said it would not comment on the criticism or whether there are any second thoughts about the joke.
Katherine Hull Fliflet, spokeswoman for the Washington-based RAINN (Rape, Abuse and Incest National Network), said she did not find the line offensive.
"I think the show is making it clear that rape is not funny by how they are positioning the joke," Fliflet said. "It's my hope that would be the viewers' take-away."
RAINN, which says it is the nation's largest anti-sexual assault organization and operates a rape hotline, works with creators in Hollywood to help them depict sexual assault realistically. The group lists actress Christina Ricci as a national spokesperson.
The National Organization for Women didn't respond to requests for comment on the Fox comedies.
MacFarlane brought up the line during a recent interview with Entertainment Weekly, predicting he will get attacked for it in the media. "But in context," he said, "it's pretty funny."
Winter said he didn't think the subject was worth joking about, and said he was particularly concerned about its exposure to younger viewers who may be fans of "The Simpsons," but are not familiar with the "Family Guy" style of comedy.
"We don't mock certain groups because we realize that it is highly insensitive and morally wrong," he said. "Why wouldn't we do the same thing about sexual assault?"