Tom Watson has hit back after his claims Marxist revolutionaries are infiltrating the party were dismissed as "nonsense" by Jeremy Corbyn.
Labour's deputy leader has published evidence on his Facebook page he claims show a hard left group is targeting young supporters of Mr Corbyn.
"I'm not making this stuff up, as others have claimed," says Mr Watson.
Labour leader Mr Corbyn in an interview with the Observer said Mr Watson's claims were not credible.
"I just ask Tom to do the maths - 300,000 people have joined the Labour party.
"At no stage in anyone's most vivid imagination are there 300,000 sectarian extremists at large in the country who have suddenly descended on the Labour Party," Mr Corbyn said.
But on Sunday night Mr Watson said in a statement: "I know Jeremy is very busy on the campaign trail and is therefore relying on others to tell him what I have said.
"To repeat again, I have never said that all our new members are Trotskyists.
"I have never claimed that hundreds of thousands of new joiners are revolutionary socialists and those who claim I did are attacking a straw man.
"I simply want to ensure that organisations like the Alliance for Workers Liberty, who have instructed all their members to join the Labour Party and target our youth sections for recruitment, are dealt with under our rules.
"It's undeniable that this is happening. AWL even published these instructions on their website."
He claims a small group of AWL activists are attempting to infiltrate the party to indoctrinate young members in "revolutionary socialist ideas".
Owen Smith, who is challenging Mr Corbyn for the Labour leadership, has steered clear of the row.
But Labour's former acting leader Dame Margaret Beckett claimed some of the party's new members did not have its best interests at heart.
"In principle everyone should be happy that more people are wanting to join the party, as long as it is the Labour Party they're wanting to join.
"We have had examples of people saying 'I want to join the Labour Party because of Jeremy but of course if he ceases to be the leader I shall leave'.
"Those are not members of the Labour Party, those are members of a fan club."
She added: "It's perfectly nice and legitimate thing to be a member of a fan club and they may get a great deal of satisfaction from it but that doesn't mean you belong to the Labour Party and I'll be sorry to think that vast numbers of those people in fact do not really want to be in the Labour Party, they just want to support Jeremy."
The leadership contest between Mr Corbyn and rival Owen Smith could split the party, she warned, adding: "Some of the people around Jeremy, (it has) unfortunately become clear in recent weeks, are perfectly happy for that to happen."