The makers of Sesame Street say characters Bert and Ernie will not marry in a same-sex ceremony despite an online petition calling for the union.
Campaigners say the best friends should marry as a way to encourage tolerance of gay people.
Nearly 7,000 have signed the petition, with more than 3,000 joining a Bert and Ernie Get Married Facebook page.
A statement from the show's makers said: "They remain puppets and do not have a sexual orientation."
But they conceded that the pair are "male characters and possess many human traits and characteristics".
The confirmed bachelors have lived together for 40 years and sleep in the same bedroom, albeit in single beds.
"Bert and Ernie are best friends," the statement from Sesame Workshop added. "They were created to teach preschoolers that people can be good friends with those who are very different from themselves.
The online petition states: "We are not asking that Sesame Street do anything crude or disrespectful," adding, "It can be done in a tasteful way. Let us teach tolerance of those that are different."
Sesame Street was created by TV producer Joan Ganz Cooney and psychologist Lloyd Morrisett in 1969 and gained a huge following, led by the creations of master puppeteer Jim Henson.
Beloved characters include Elmo, the Cookie Monster and Big Bird.
It is still watched in more than 140 countries and averages 120 million viewers worldwide.