Boris Johnson has said it would be "hypocritical" for US president Barack Obama to seek to use his upcoming visit to the UK to try to persuade Britain to stay in the European Union.
The White House has indicated that Mr Obama - who has previously voiced support for continued UK membership - is ready to offer his view as a "friend" if asked about Brexit during his two-day visit, though he will stress that it is a decision for the British people.
The president is due to have lunch with the Queen at Windsor Castle before talks with David Cameron in 10 Downing Street on April 22, and will answer questions from members of the public at a "town hall" event in London the following day.
But Mr Johnson said the US should not urge Britain to stick with a position it would not accept for itself, telling the Evening Standard: "I honestly don't mind the idea of him joining the debate.
"Where we do part company, and where I do mind, is that it is plainly hypocritical for America to urge us to sacrifice control - of our laws, our sovereignty, our money and our democracy - when they would not dream of ever doing the same."
The London Mayor was leading a "Brexit blitz" on the first day of the official campaign for the June 23 referendum, with an address in Manchester, ahead of weekend events in Leeds and Newcastle.
And the Remain camp also wheeled out a big gun, with former chancellor Lord (Alistair) Darling accusing Brexit backers of offering "Project Fantasy", as he warned that leaving would threaten Britain's economy.
Meanwhile, Ukip leader Nigel Farage challenged Mr Cameron to a one-on-one debate on the claims contained in a leaflet sent out to households around the country, setting out the Government case for continued EU membership.
Handing his own copy of the document back to 10 Downing Street, Mr Farage also took a swipe at Mr Obama, branding him "the most anti-British American president there has ever been".
The Vote Leave organisation, backed by Mr Johnson and Cabinet heavyweight Michael Gove, moved the NHS centre stage in the campaign as it claimed a large chunk of the UK's £10.6 billion net contribution to Brussels could be diverted to medical care if Britain quits the EU.
Unveiling the campaign's first billboard ad, Labour MP Graham Stringer said: "Our NHS is struggling to cope with rising demand and needs the support that is currently siphoned off to Brussels. Instead of handing over £350 million a week to the EU we should spend our money on our priorities like the NHS."
But Downing Street insisted that Brexit would mean "less money for the NHS", with Mr Cameron's official spokeswoman saying: "A strong NHS needs a strong economy and the Prime Minister has been very clear that our economy is stronger in the EU."
Health unions blasted Vote Leave's figures as "spurious and outrageously misleading", insisting the NHS's financial woes were made in Whitehall and not Brussels.
Unite national officer for health Barrie Brown said: "It defies belief to think that Boris Johnson and Michael Gove would do a massive political U-turn and divert billions of EU cash into the NHS - when they have supported real cuts to the NHS budget and been enthusiastic flag-wavers for the privatisation and break-up of the NHS."
And TUC general secretary Frances O'Grady said: "The reality is that Brexit would plunge the NHS into a staffing crisis, which could lead to the longest hospital waiting lists we've ever known. And with experts warning that Brexit would hit Britain's economy, the consequences for NHS funding would be dire."
Justice Secretary Mr Gove is expected to echo the NHS warnings during a speech in Nottingham, as fellow leading Leave figures Commons Leader Chris Grayling and Northern Ireland Secretary Theresa Villiers also step up the Brexit campaign in the coming days.
In a speech in London, Lord Darling acknowledged that either side could win the EU contest.
"This is a very, very close vote. No one can predict with any certainty what is likely to happen," said the Labour peer.
"I hope we will win and I hope we will win well but we need to get the support of people the length and breadth of the country, no matter what their political allegiance has been in the past.
"We need every single vote. It is going to be very close. Every vote counts."