Leave campaigners point out two-thirds of FTSE 100 firms, including Tesco and Sainsbury, did not back the letter.
A referendum on whether the UK should stay in the EU will be held on 23 June.
In a move described by No 10 as "unprecedented", chairmen or chief executives of 36 FTSE 100 companies signed the letter backing the campaign to stay in the EU, including Burberry, BAE Systems and EasyJet.
The FTSE bosses were among a total of 198 signatories from the business world, including the chief executives of Heathrow and Gatwick airports.
However, nearly two-thirds of the UK`s largest publicly listed businesses did not sign and the BBC`s economics editor Kamal Ahmed said there were some notable absentees among them, such as the bosses of Tesco, Sainsburys, RBS and Barclays.
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