Such protests occur every year outside the former embassy compound to mark the anniversary of its 1979 takeover following the Islamic Revolution. But Monday's rally is the largest in years after calls by groups such as the Revolutionary Guard for a major showing, including chants of "death to America" that some of Rohani's backers have urged halted.
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The fact that Iranian hardliners demonstrated to mark the anniversary serves as a sign of the considerable obstacles to Rohani's diplomatic initiative to ease tension with Washington faces.
Despite the fact that Rohani's overtures are backed by Iran's Supreme Leader Ayatollah Ali Khamenei, opponents say they will not back down, raising the prospect of deeper internal rifts.
Large crowds gathered early on Monday morning around the embassy building – often described as the "nest of spies" – holding anti-US placards, waving flags and shouting "Death to America", Iranian media reported.
The 1979 siege began when student activists stormed the embassy, taking hostage 52 embassy staff for 444 days. The two countries have not re-established diplomatic relations since.
While the rally is an annual event, this year has taken on greater significance because of the new government's attempts to ease tensions with Western countries exacerbated during the two terms in office of the previous president, hardliner Mahmoud Ahmadinejad.
Rohani was elected in June on a platform of establishing better diplomatic relations and jump-starting talks over Iran's controversial nuclear program.
A short telephone conversation between him and US President Barack Obama after the UN general assembly in September was applauded by many Iranians but met with suspicion by hardline Iranian groups.
On Sunday, the Islamic Republic's most powerful figure, Supreme Leader Ayatollah Ali Khamenei, gave strong backing to Iran's nuclear negotiators, an apparent warning to hardliners against accusing Rohaniof compromising with its old enemy.
"No one should consider our negotiators as compromisers," Khamenei said in a speech. "They have a difficult mission and no one must weaken an official who is busy with work."
On Saturday, an editorial by conservative newspaper Kayhan warned against trusting the United States in current nuclear negotiations and said there were signs that "the Americans are aiming to trick the Islamic Republic" in the next round of talks this week.
The new mood of Iranian diplomacy has also brought into question the use of the slogan "Death to America". While moderate figures have suggested it is time to drop the phrase, conservatives say it is as important now as ever before.
In an interview with Arabic-language state TV channel, the head of the parliamentary committee for national security, Alaeddin Boroujerdi described the chant as the "mildest response" to America's actions of interfering in countries around the world.
Reuters and the Associated Press contributed to this report
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