Dozens of lawmakers led a spontaneous demonstration in parliament, pumping their fists in the air while demanding the execution of Mir Hossein Mousavi and Mehdi Karroubi, who have been under house arrest since last week.
"We believe the people have lost their patience and demand capital punishment," more than 200 lawmakers said in a statement.
Last year, Iran's leaders tolerated a flurry of statements by the two former presidential challengers turned opposition leaders, and in turn, neither called for any demonstrations. But the anti-government protest that rocked the center of Tehran on Monday signaled an end of that unspoken arrangement, analysts say.
"The efforts of the Supreme Leader were focused on trying to bring Mousavi and Karroubi back into the folds of the revolution," Kazem Jalali, a member of the National Security and Foreign Policy Commission of the parliament, told the semiofficial Fars News agency. "But these persons have purged themselves from the system. The parliament demands the strongest punishment for Mousavi and Karroubi."
Although government supporters have often called for both men to be arrested, it is rare for higher officials to demand their execution.
Ahmad Khatami, an influential Friday prayer leader appointed by Khamenei, joined in by saying the opposition leaders were guilty of "moharebeh," a Shiite legal term that means "waging war against god" and carries the death sentence in Iran.
The paramilitary Basij organization, which has been instrumental in cracking down on the protests, also issued a a statement asking authorities to "swiftly change their decision."
The demands pose a dilemma for Supreme leader Khamenei, who has final say in all important state matters. During the past year, he effectively prevented Karroubi and Mousavi from being arrested by suggesting on several occasions that the two men must admit their mistakes and return to the embrace of Iran's Islamic system.
Following his guidance, members of Iran's National Security Council, which runs many day to day affairs and is headed by President Mahmoud Ahmadinejad, concluded that their arrest or punishment would turn them into living martyrs for the opposition movement and could incite new protests, many here say.
But the mood has dramatically changed since Monday's demonstration, in which two people were killed, according to Fars. Although opposition Web sites have said the men were protesters, state organizations reported that they were members of the Basij and were killed by the opposition.
A state-backed funeral procession is planned on Wednesday for one of the men, named Sal-e Jhale, a 26-year-old ethnic Kurd, the semi-official Mehr news agency reported.
"The use of firearms in these riots is a declaration of armed war against the Iranian nation ... this should be seen by the officials," the Basij organization said in a statement posted online by Fars.
Although there were no protests on Tuesday, the Internet remains restricted.
Monday's events have increased demands for legal action against Karroubi and Mousavi, said Amir Mohebbian, an analyst close to Iran's ruling class. "The leader might now change his view on their arrest, although nothing is certain just yet," he said.
Ahmadinejad, speaking on state television during a scheduled live broadcast, said the demonstrators and leaders had planned to tarnish the "brilliance" of the Iranian nation.
"The Iranian nation is a shining sun. They [the demonstrators] threw some dust towards the sun," he said. "I want to address those who planned and masterminded this protest. They should know that the dust will certainly return into their own eyes."