Downing Street refused to deny this morning that Number 10 officials contacted the British Chambers of Commerce (BCC) over former director general John Longworth's euroscepticism.
"Of course Number 10 talks to business organisations regularly. But if you are referring to Mr Longworth’s decision to step down, that is entirely a matter for the BCC," the Prime Minister's spokeswoman told journalists today.
"We should be absolutely clear that no pressure was applied by Number 10," the spokeswoman added. "This is a decision for Mr Longworth and the BCC. The president of the BCC has set out very clearly the reason behind that decision."
The BCC said last night that Longworth had stepped down as director general of the business group with immediate effect.
Longworth was first suspended from the role last week for saying Britain would be better off outside of the European Union.
"With the reforms that we have received so far, the UK would be better off taking a decision to leave the European Union," Longworth told Sky News on Thursday, the same day as the BCC's annual conference.
The BCC said last night that Longworth's resignation was the result of a mutual agreement between the former director general and the group's board "and there were no external factors involved".
The organisation said it recognised that Longworth's "personal view on the referendum is likely to create confusion regarding the BCC’s neutral stance going forward".
The BCC has repeatedly said it will not campaign for either side in the run-up to the EU referendum on 23 June.
A recent BCC survey of its members found that 60 per cent would vote to remain in the EU, while only 30 per cent would vote to leave.
Longworth's suspension and resignation have raised repeated questions over whether the government had intervened in the matter.
London mayor Boris Johnson, who backs Brexit, called Longworth’s suspension “scandalous” over the weekend, saying the business boss had been "crushed by the agents of ‘project fear’" – a thinly-veiled reference to pro-EU campaigners.
Longworth did not rule out government interference in interviews today, telling Sky News: "What I can say is, of course, that government departments, including Number 10, from my experience, actually contact business representatives all the time and express their views, sometimes very strong views and strident views.
"But I have to say in my experience it has never affected my judgement," Longworth added. "I have always represented business views without fear or favour."
In a separate interview with the Telegraph, Longworth accused the government of trying to "scare" voters into staying in the European Union, saying: "It is highly irresponsible of the government of the country to be peddling hyperbole."
"It is alright for the campaign groups to do it because they are promoting a particular position. But the government has to be responsible."
Meanwhile, Conservative MP David Davis today launched a Freedom of Information (FOI) request for details fo all government communication with the BCC relating to Longworth's suspension and resignation.
Davis, a spokesman for the Grassroots Out eurosceptic campaign, said: "The last thing we want to see is a witch-hunt against business leaders brave and astute enough to make the argument that Britain would be better off economically if it regained the power to strike its own trade deals and was freed of the crippling burden of red tape, costing many billions a year, imposed by Brussels.
"We need a clear statement by 10 Downing Street that the government was not involved in pressurising the BCC into suspending Mr Longworth. We need to know there was no contact between ministers, officials or advisers and the BCC before Friday’s BCC board meeting that took the decision to suspend its chief."