The Prime Minister has accused Boris Johnson of “getting it wrong” on the EU and said he hoped for a “civilised and reasonable”.
David Cameron said he had “great respect” for the London Mayor and that he had a “very strong future in British politics” but added “on this issue I think he has got it wrong”.
It comes after he launched a stinging attack on Mr Johnson in the House of Commons calling the mayor”s suggestion of a second referendum on the UK”s membership of the EU “for the birds”.
Mr Cameron was speaking as he made his case to remain in the EU to voters for the first time at an event at a Slough business.
It came after Mr Johnson denied that the EU would split the Conservative Party and that the Tories would unite behind Mr Cameron after the EU referendum, despite signs of a deepening rift in the party over Europe.
Former Foreign Secretary Lord Hague warned the party it could lose the next election if it became embroiled in in-fighting over the EU.
Speaking on Tuesday morning, Mr Johnson said: “I think one of the big differences between now and what I remember back in the 1990s is that the Conservative Party actually has a much better team spirit, much better feeling about this stuff.
“I”m sure, I have absolutely no doubt that after this is over the Tory Party is going to unite again around David Cameron”s leadership.”
Mr Cameron told people at the Slough event that if the UK hadn”t been in the EU then one of the 21/7 terror attack plotters would not have been captured and jailed. He said the UK was safer to remain in.
And he said that businesses were better off in the European Union, highlighting the support from business leaders in a letter published on Tuesday morning.
Mr Johnson also criticised the letter signed by 200 top business leaders warning leaving the EU would “deter investment and threaten jobs”.
He accused them of “supporting alarm and anxiety”.
Chairmen or chief executives at 36 FTSE 100 companies – including BT, Marks & Spencer, Kingfisher and Vodafone – were among the signatories.
Leader of the House of Commons Chris Grayling, who is to campaign against the Prime Minister, said big businesses had called on the UK to adopt the Euro and had it done so that would have been a “disaster”.
He said: “It is also very noticeable that some of our biggest manufacturers – Toyota, Honda, Nissan – all said that it would make no difference to their plans at all if we were to leave the European Union.”
As first reported by Sky News City Editor Mark Kleinman, some of the PM”s closest business advisers refused to sign the letter.
Members of Mr Cameron”s Business Advisory Group (BAG) who opted not to sign include Alison Brittain, chief executive of Whitbread; Jeff Fairburn, who runs the housebuilder Persimmon; Liv Garfield, the Severn Trent boss; Robert Noel, Land Securities” chief executive; Steve Varley, the UK chairman of EY; and Nigel Wilson, chief executive of Legal & General.
Tesco, Sainsbury”s and Barclays are also absent, but the chief executives of Heathrow and Gatwick airports have signed up.
The letter says: “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 release of the letter is a repeat of the letter from business leaders used successfully during the campaign for Scotland to stay in the Union.