The challenge was sparked by the resignation of Nationals deputy leader Bridget McKenzie on Sunday over her allocation of $100 million in sports grants, which opened the position of deputy leader.

In the turmoil the party feared as pressure mounted against Senator McKenzie, disgruntled members of the 21-strong parliamentary party jumped on the opportunity to challenge Michael McCormack, whose low-key style has brought criticism since he took over as leader from Mr Joyce. Mr Joyce was forced to resign in February 2018 after an affair with his staffer, who became his partner.

Queensland MP Llew O'Brien made the decisive move late on Monday, confirming he would move a leadership spill on Tuesday. Mr O'Brien did not declare his support for Mr Joyce, leaving open the possibility of a third contender.

But at 7pm Senator Canavan, now the Nationals' most senior Senator, threw his support behind Mr Joyce after insisting as late as 6.45pm that Mr McCormack had his full support.

"We need a bulldog, we need a fighter," he said.

He also revealed his own membership of a sports club that received funding under a program in his portfolio.

David Littleproud was top contender for the deputy's position, but his office said he would not stand for leader.

"Nothing has changed for David, he only ever made himself available for the deputy leadership," a spokeswoman said after the spill was announced.

It was a day of upheaval, with the Greens also leaderless after Richard Di Natale's resignation on Monday.

The Greens appear likely to appoint former lawyer and co-deputy leader Adam Bandt as leader on Tuesday, with Mr Bandt confirming his candidacy.

Senator Di Natale leaves after a decade in parliament to spend more time with his family, including sons aged 9 and 11, and to support wife Lucy Quarterman's career. Senator Di Natale said having decided he would not stand at the next election, he had concluded over the summer that now was the time to leave.

Throwing more leadership mayhem into the mix, maverick Queenslander MP Bob Katter, stood down as leader of his own party, saying he would stay in parliament but his son, state MP Robbie Katter, would become leader of Katter's Australian Party.

Mr Joyce said the Nationals needed to get on the front foot.

"The National Party has to be on the balls of its toes as we face one of the most challenging times and we have to speak with our own voice," he said.

Mr McCormack did not respond to the news of the spill. Earlier in the day, he insisted there was no vacancy for the leader's job and he did not expect a spill.

"I am the leader of the party, I have the party's support," he said. "People out in regional Australia watching this telecast or reading newspapers in recent days, they're not focused on the machinations of the National Party."

Backbencher David Gillespie declared his candidacy for the deputy's role, and was the strongest public critic of Mr McCormack's performance on Monday. He praised Mr Joyce's popularity.

"Regional Australia is crying out for more transparent vocal representatives in the leadership role," he told Sky News. "Michael does a lot of media in the bush, but when you're in the leadership I would have liked him to have done - and I would still like him to do - more in the national space. People in my area want to see their leaders out and about."

Mr Littleproud made no public comment.

Matt Canavan, now the Nationals' most senior Senator, ruled himself out for deputy or leader, and said Mr McCormack had his full support has leader.

Queenslander Keith Pitt would also seek the deputy's job, the ABC reported.

Senator McKenzie herself would not say who she would support as deputy, but said, "In true National Party fashion, I'm sure there will be a Melbourne Cup field of candidates."

Senator Sam McMahon said earlier that while a number of names had been suggested in the media as deputy contenders, she had been lobbied by just two - Mr Littleproud and Mr Gillespie. She had not spoken with Mr Joyce and had no direct knowledge of a challenge, she said, speaking after 5pm.

"Given that I'm reasonably new to this I'm not going to stick my neck out on it. As far as I know there is only one vote tomorrow and that's for deputy," she said.

Michelle Landry backed Mr McCormack.