In his statement to Iran, which left no room for interpretation, Franco Frattini said that unless a reply was forthcoming by the end of the day, "it would implicitly mean that they have declined."
But a spokesman for Iranian Foreign Minister, Manouchehr Mottaki said the decision would not be made until Friday, which is the end of the Iranian week.
Italy, current holder of the G8 presidency, invited Iran to attend the meeting which will focus on ways to improve stability in Afghanistan and Pakistan.
But on Monday, the Italian news agency, ANSA, quoted Frattini as saying he did not know what kind of contribution Iran could make given its troubled domestic situation. He said it was "simply a matter of being concrete and pragmatic."
He joined in the international choir calling for Iran to stop the crackdown against demonstrators protesting the re-election of President Mahmoud Ahmadinejad. "What we ask above all is a halt to the violence, that there are no more dead or people attacked in the streets."
Calls for election review
Frattini also echoed German Chancellor Angela Merkel's urgings for a review of the disputed presidential elections.
In a statement on Sunday, Merkel called for a recount. She said Germany "is on the side of the people of Iran who want to exercise their right to freedom of expression and free assembly."
She also appealed to the leadership in Iran to permit peaceful demonstrations, refrain from using force against protestors and release imprisoned opposition figures.
"Human and civil rights have to be fully respected," she said.
For its part, the Iranian government has said it is reviewing relations with Germany, France, Britain and the United States over their stance towards the contested presidential elections.
A German foreign ministry spokesman said on Monday that Berlin had 'invited', but not summoned, Iran's ambassador to clarify the statements.
Meanwhile the EU foreign policy chief, Javier Solana has criticized Iran's attempts to muzzle journalists working there. Speaking in response to the expulsion of the BBC's permanent correspondent in Tehran and the detention of other reporters, Solana said it was "not something we can accept."
On Monday, spokesman for Iran's foreign ministry, Hassan Ghashghavi blamed the BBC and Voice of America for the unrest in Tehran. He said their aim was to "weaken national solidarity, threaten territorial integrity and disintegrate Iran."
He added that Iran was currently considering the expulsion of foreign diplomats. "The foreign minister will consult today with parliament's foreign policy commission about the exact dimensions of interference," he said.
Speaking in Brussels on Monday, Swedish Foreign Minister Carl Bildt said a summoning of EU ambassadors by Tehran would be a sign that they were being heard. "From that perspective," he said, it would be "good."
Bildt, whose country takes over the rotating EU presidency on July 1, also said it was up to Tehran to "establish the credibility of their political process."
Editor: Trinity Hartman