The UK has told an Iranian diplomat that Ayatollah Khamenei's description of the British government as "evil" is unacceptable.
UK officials had summoned Iran's ambassador, Rasul Movaheddian, to the Foreign Office to lodge a protest.
But they were told he was unavailable and Iran sent its charge d'affaires, a more junior official, in his place.
The row was sparked by Ayatollah Khamenei saying the UK was the "most evil" of Western governments.
A Foreign Office spokesman said: "We made clear to the Iranian charge that the supreme leader's comments were unacceptable and not based in fact. In the ambassador's absence, the charge was called in."
Shift of position
The meeting at the Foreign Office is understood to have lasted less than 20 minutes.
The BBC News website world affairs correspondent, Paul Reynolds, says the decision to summon senior Iranian diplomats represents a shift of position by the British government which up until now had wanted to avoid getting involved in public arguments with Iran.
IRAN ANALYSISHe added: "The line had been that it wanted to avoid giving the Iranians any reason to blame Britain for interfering. The US government has taken a similar view.
"However, Ayatollah Khamenei's description of Britain as the most 'evil' of foreign governments was a step too far."
British diplomats are thought to believe Britain is being used as "proxy" for the United States, because Iran does not want to endanger its improving relations with America.
In his first public remarks after days of demonstrations, Ayatollah Khamenei issued a stern warning that protests against the country's disputed presidential election results must end.
In his address to tens of thousands of worshippers at Tehran Friday prayers, which was broadcast live by Iranian state TV and radio, he said the outcome must be decided at the ballot box, not on the street. He voiced support for President Mahmoud Ahmadinejad, saying the president's views on foreign affairs and social issues were close to his.
Demonstrators calling for a new election earlier vowed to stage fresh protests on Saturday.
Ayatollah Khamenei also lashed out at Western governments in his address.
He told worshippers: "I urge old friends and brothers to be patient and keep control of ourselves.
"Please see the hands of the enemy. Please see the hungry wolves in ambush, who are gradually even removing their mask of diplomacy and showing their true faces. Be aware of them.
"Today, senior diplomats of some Western countries, who addressed us diplomatically up until today, have now removed their masks. They are showing their true faces.
"They are showing their enmity against the Islamic Republic system and the most evil of them is the British government."
'Condemning violence'
Speaking at the European summit in Brussels, UK Prime Minister Gordon Brown insisted that Britain and the EU wished to have good relations with Iran, but that human rights should be respected.
He said: "The whole world is looking at Iran, at the speech that has been made today but also what is happening in Iran has been happening over the last few weeks.
"It is for the Iranian people to decide their future in elections.
"We are with others, including the whole of the European Union, unanimous today, in condemning the use of violence, in condemning media suppression and in condemning attempts, of course, to make sure that there are people, who are political prisoners who are not free to express their views in Iran.
"What we want is to have a good relationship with Iran in the future but that depends on Iran being able to show to the world that its elections have been conducted fairly and that there is no unfair suppression of rights or of individuals in that country."
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