"The allegations against Congressman Conyers, as we have learned more since Sunday, are serious, disappointing and very credible," Pelosi said during a briefing in the Capitol.
"It's very sad. The brave women who have come forward are owed justice. I pray for Congressman Conyers and his family, and wish them well. However, Congressman Conyers should resign," Pelosi said.
Moments later, Ryan also came forward asking for Conyers to resign.
"This needs to be a workplace where everyone feels safe and respected," Ryan said. "No woman should have to endure harassment in any form in any institution, let alone here in Congress.
This comes as Conyers was hospitalized in Detroit on Thursday.
Sam Riddle, a family friend of Conyers, said the Michigan Democrat is "resting comfortably" with his wife at the hospital, but did not give a reason why he was admitted.
"John Conyers is doing okay, as well as can be expected for a gentleman that's approaching 90 years of age," Riddle said. "You can wait out here all you want; he won't be coming out."
The 88-year-old congressman returned to Detroit this week after sexual harassment accusations continued to pile up from former staffers.
One of the women, Marion Brown, spoke to NBC's Today.
"Some of the things that he did, it was sexual harassment," Brown said. "Violating my body, propositioning me, inviting me to hotels with the guise of discussing business, and then propositioning for sex. He just violated my body. He has touched me in different ways, and it was very uncomfortable and very unprofessional."
Conyers stepped down from his position as the ranking Democrat on the House Judiciary Committee but denied the allegations.
"Many of [the accusations] were raised by documents reportedly paid for by a partisan alt-right blogger." Conyers tweeted. "I very much look forward to vindicating myself and my family before the House Committee on Ethics."
Riddle said Conyer's health issues were stress-related from recent media attacks that Conyers was "used to" from "serial attackers."
"The congressman's health is not what it should be and lot of that is directly attributable to this media assault," he said.