Top executives at Asda, BT, Marks & Spencer, Kingfisher and Vodafone 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” and called on Britain to remain in the EU to help growth.
In a letter to The Times, the bosses 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.”
But letter includes notable absences, such as Tesco, Sainsbury”s and Barclays.
Among the signatories are Tory donors and figures who have accepted Government roles under David Cameron”s premiership.
Roland Rudd, treasurer of Britain Stronger in Europe, which organised the letter with Downing Street’s support, said: “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.”
The Prime Minister was forced to defend using a Number 10 civil servant to lobby businesses to support the pro-EU campaign after MPs heard the letter was about to be published.
He said: ”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.”
The support from some of Britain”s most renowned businesses will be welcomed in Downing Street, as a growing number of Conservatives declared they would be backing the leave campaign.
The Prime Minister made his displeasure at Boris Johnson”s decision to back a Brexit clear in a scathing attack on the London mayor.
In front of packed Commons chamber, the Prime Minister said his own pledge to step down at the general election meant he had “no agenda” other than the interests of Britain – a dig at Mr Johnson”s 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.