Alabama Senate candidate Roy Moore "should step aside", House of Representatives Speaker Paul Ryan has said.
Mr Moore is facing claims of sexual misconduct and Mr Ryan described the allegations as "credible".
Mr Ryan joined the growing chorus of Washington Republicans calling on Mr Moore to quit the race after two women stepped forward describing how he groped them when they teenagers decades ago.
Republican candidate Mr Moore has called the reports false.
Senate Republicans, led by Majority Leader Mitch McConnell, have been struggling to find a way to force Mr Moore out.
His name remains on the ballot for the December 12 special election against Doug Jones.
The latest accusations came when Beverley Young Nelson claimed she was sexually assaulted by Mr Moore in a locked car when she was a teenager in the 1970s.
In tears, Ms Nelson said that when she was 16, Mr Moore offered her a ride home from the restaurant where she worked.
She accused him of touching her breasts and locking the door to keep her inside his car.
She said he squeezed her neck while trying to push her head towards his crotch and tried to pull her shirt off.
Mr Moore’s campaign released a statement saying that lawyer Gloria Allred - representing Ms Nelson - "is a sensationalist leading a witch hunt".
It said Mr Moore is innocent and "has never had any sexual misconduct with anyone".
Mr Moore abruptly called a press briefing in Gallant, Alabama, following Ms Nelson’s news conference in New York.
"I can tell you without hesitation this is absolutely false. I never did what she said I did. I don’t even know the woman," Moore said.
He signalled he had no intention of ending his candidacy, calling the latest charges a "political manoeuvre" and launching a fundraising appeal to "God-fearing conservatives" to counter his abandonment by Washington Republicans.