The chancellor and health secretary have resigned from government, saying they no longer have confidence in Boris Johnson to lead the country.
Chancellor Rishi Sunak said the public expected government to be conducted “properly, competently and seriously”.
Health Secretary Sajid Javid echoed this, saying the government was not “acting in the national interest”.
The resignations came minutes after the PM apologised for appointing MP Chris Pincher to a government role.
Mr Johnson admitted he had made a “bad mistake” in appointing Mr Pincher to the role of deputy chief whip earlier this year, despite being told about earlier allegations about the MP’s conduct.
His handling of the row has come in for fierce criticism from the opposition and some of his own MPs.
But the BBC understands Foreign Secretary Liz Truss, Levelling Up Secretary Michael Gove and other cabinet ministers are backing the prime minister as he assesses the scale of the rebellion against his leadership.
Asked if that was an error to appoint Mr Pincher, Mr Johnson said: “I think it was a mistake and I apologise for it. In hindsight it was the wrong thing to do.
“I apologise to everybody who has been badly affected by it.”
The resignations of two senior cabinet ministers plunge Mr Johnson into a fresh leadership crisis weeks after he survived a no-confidence vote.
Mr Johnson won the backing of a majority of Tory MPs in the vote despite a significant revolt against his leadership.
The PM won 59% of the vote, meaning he is now immune from a Conservative leadership challenge until June next year under party rules.
Labour leader Sir Keir Starmer said cabinet ministers should act in the national interest to remove Mr Johnson from office, urging them to resign, or force him to do so.
“They know what he’s like,” Sir Keir said. “He’s said that he’s psychologically incapable of changing, and therefore they have to do what’s in the national interest and remove him.”
Is this the beginning of the end for Boris Johnson?
During the course of the day, some of the PM’s prominent critics called on government ministers to put pressure on the prime minister to resign.
A chancellor, Rishi Sunak, and an ex-chancellor, Sajid Javid, have done just that.
Both men see their resignations as necessary if the PM is to be pushed out.
Both may be positioning for a future leadership contest.
But even now, Downing Street will hope to avoid this.
Boris Johnson still has his foreign secretary, home secretary, defence secretary and business secretary.
And, remember, a beleaguered Gordon Brown survived a ministerial resignation because the rest of his cabinet stayed loyal, when he was in Number 10.
But it now seems more likely that other ministers, in more junior roles, who have been privately critical of Boris Johnson could follow Mr Sunak and Mr Javid’s lead.
Mr Johnson’s government has been dogged by a series of controversies since the Conservatives won a landslide election victory in 2019.
Discontent among Tory MPs has grown since a highly critical report into lockdown parties in and near Downing Street during the Covid-19 pandemic was published earlier this year.
The report laid bare the extent of Covid rule-breaking in Number 10, including at a birthday party Mr Johnson was fined by the police for attending in June 2020.
The fine meant Mr Johnson became the UK’s first serving prime minister to be sanctioned for breaking the law.
Some Tory MPs have also expressed dissent over tax rises, the government’s response to rising living costs and its policy direction.