The drama, based on the scandalous lives of New York teenagers, has courted controversy since it first aired in 2007, with storylines featuring drugs and sex.
But the show, starring Blake Lively and Leighton Meester, has now provoked a storm after programme bosses hinted an upcoming episode will feature a three-way sex scene.
The promo for the episode convinced officials at leading campaign group the Parents Television Council (PTC) to write to bosses at the CW Network, which airs the show, urging them to ditch the controversial footage.
PTC president Tim Winter says in a statement, "(This show is) expressly targeted to impressionable teenagers. Will you now be complicit in establishing a precedent and expectation that teenagers should engage in behaviours heretofore associated primarily with adult films?
"If this programme violates broadcast decency law, rest assured that our members will contact the Federal Communications Commission."
The episode in question is expected to air in the U.S. on 9 November (09).