Peter Dutton has told Malcolm Turnbull he has the majority support of the Liberal party room and wants to challenge him again.
The ex-Home Affairs Minister lost a challenge 48 votes to 35 on Tuesday but now wants to have another go at toppling his leader.
"A few minutes ago I spoke with Malcolm Turnbull to advise him I believed the majority of the party room no longer supported his leadership," Mr Dutton tweeted this morning.
"Accordingly, I asked him to convene a party room meeting at which I would challenge for the leadership of the Parliamentary Liberal Party."
Two more federal government ministers have formally resigned to support Mr Dutton's bid.
Assistant ministers Michael Sukkar and Zed Seselja have refused the prime minister's offer to remain on in their positions, throwing their weight behind Mr Dutton ahead of a second leadership challenge.
The pair have followed Mr Dutton, Concetta Fierravanti-Wells and James McGrath from the front bench.
Mr Sukkar said the prime minister had asked him to provide "unequivocal support" for him in any future ballots after voting against him in Tuesday's leadership spill.
"I am unwilling to give you this commitment, and therefore formally tender my resignation," he wrote in a letter to Mr Turnbull on Thursday.
Meanwhile, Nationals MP Kevin Hogan has threatened to quit the government and sit on the cross bench if there is another Liberal leadership spill.
Mr Hogan, from northern NSW, says his community is fed up with the constant leadership changes over the past 10 years.
"I will support the government in no-confidence motions and supply. Any other legislation I will take on a case-by-case basis," he said on Thursday.
A small group of Liberal MPs are pushing Mr Dutton, the former home affairs minister, to launch a fresh challenge to Mr Turnbull's leadership.
But new polling shows a Dutton government would crash at the election to Bill Shorten, with voters picking the Labor leader over the former home affairs minister.
A defiant prime minister, who has arrived at Parliament House, is holding on to his leadership with support from his two key lieutenants, Treasurer Scott Morrison and Finance Minister Mathias Cormann.
Mr Dutton's supporters circulated a letter on Wednesday night calling for the party room to meet.
It was unclear how many MPs had actually seen and signed the letter and if any cabinet ministers were willing to shift their support from Mr Turnbull.
Mr Dutton admitted he's calling Liberals to win support for a second challenge after failing 48 votes to 35 in a snap leadership ballot on Tuesday.
The electorate, however, appears to be supporting Mr Turnbull.
A Morgan poll of more than 1200 voters picked Mr Turnbull as the better prime minister over Mr Shorten, 52 per cent to 44.5 per cent.
But Mr Shorten thumped Mr Dutton 59 per cent to 36.5 per cent when voters were given the chance to pick between them.
Mr Dutton launched a media campaign on Wednesday morning, going on Melbourne radio to call for a royal commission into fuel and energy prices.
But Mr Morrison unleashed on Mr Dutton's plan to take the GST off electricity prices, calling it a "budget blower" that would cost $7.5 billion over four years.
Mr Dutton's push for another challenge lost some steam amid questions about his parliamentary eligibility over the public funding of childcare centres held under a family trust.
"The constitution is clear: you can't be taking cash for your business from the government and at the same time be a member of parliament," senior opposition MP Tony Burke told Sky News.
Former prime minister Kevin Rudd on Thursday tweeted Mr Dutton did not deserve to lead the country for boycotting the apology to first Australian.
Thursday is the last day of parliament until September 10, with the next scheduled Liberal partyroom meeting on September 11.