Bosses at more than a third of Britain's biggest businesses have come out in support of the campaign for the country to remain in the European Union.
Asda, BT, Marks & Spencer, Kingfisher and Vodafone chiefs backed a letter warning of the risks to the economy of quitting the 28-member bloc.
Chairmen or chief executives of 36 FTSE 100 companies said a Brexit would "deter investment and threaten jobs" but the total number of falls short of the 80 or 50 that it had previously been suggested would sign.
In a letter to The Times, they wrote: "Business needs unrestricted access to the European market of 500 million people in order to continue to grow, invest and create jobs.
"We believe that leaving the EU would deter investment and threaten jobs. It would put the economy at risk."
The letter includes some notable absences, such as Tesco, Sainsbury's and Barclays but the chief executives of Heathrow and Gatwick have signed up.
Among the signatories are Tory donors and figures who have accepted government roles under David Cameron's premiership, according to The Times.
Roland Rudd, treasurer of Britain Stronger in Europe, which organised the letter with No 10's support, told the newspaper: "This is the single biggest number of business leaders who have been willing to support staying in a reformed EU.
"What is also striking is the number who have done so on behalf of their companies as well as in a personal capacity."
David Cameron was forced to defend using a Downing Street civil servant to lobby businesses to support the pro-EU campaign after MPs heard the letter was about to be published.
"The Government's view is that we should remain in a reformed European Union and the civil service is able to support the Government in that role," he said.
The support from some of Britain's most well-known businesses will be welcomed in Downing Street as a growing number of Conservatives declared they would be backing the leave campaign.
David Cameron made his displeasure at Boris Johnson's decision to back Brexit crystal clear in a stinging attack on the London mayor.
In front of packed Commons chamber, the Prime Minister said his own pledge to step down at the UK general election meant he had "no agenda" other than the interests of Britain, a clear dig at Mr Johnson's barely-disguised leadership ambitions.
Eurosceptic Tories admitted Mr Cameron had left the mayor with a "bruise" and, at a meeting of backbenchers, told the premier to "be kind" to him.
Mr Cameron will hit the campaign trail later to push the case for staying in the EU during a visit to a business.
Meanwhile, Alan Johnson, who is leading Labour's pro-EU campaign, will give a speech at Airbus in Bristol warning that around 50,000 manufacturing apprenticeships in the UK are dependent on exports to the EU and could be put at risk if the UK by Brexit.
Vote Leave chief executive Matthew Elliott said: "Panic seems to be spreading in Number 10 as support for David Cameron's deal plummets.
"The EU treaties will only change by 1% if it ever comes into force - there is very little reform on the table.
"The only way to take back control of our economy to help British businesses to flourish is to Vote Leave."
Leave.EU co-founder Richard Tice said: "We remember well how many large businesses and EU funded groups like the CBI said we should join the euro. How wrong they were."
He added: "The truth is that despite the bullying of a prime minister who has no real business experience, it is other normal commercial factors which will determine the continued success of British businesses to invest and grow. Brexit will reduce unnecessary regulatory burdens and cost on business, which can be used to invest in more jobs, not less."