Reuters reported that marchers clashed with supporters of President Ahmadinejad riding motorcycles.
The BBC's Jon Leyne, in Tehran, says he understands plain-clothed militias have been authorised to use live ammunition for the first time.
Ahead of the rally, correspondents said life appeared to be returning to normal in central Tehran. Shops were open and there was little evidence of the weekend's unrest.
However, there was a small demonstration attended by Mr Mousavi's wife Zahra Rahnavard at Tehran University.
She said the campaign was continuing, and urged people to go onto their roofs shouting "God is great".
Reports said paramilitaries were trying to break up the demonstration, and that students had taken cover in a mosque.
The campus has been a focus of unrest in recent days. Overnight police and militias raided dormitories, arresting dozens of students.
In related developments:
President Ahmadinejad reportedly cancelled a trip to Moscow, where he was due to attend a regional summit on Monday
Reports say Mr Mousavi's newspaper Kalameh Sabz has suspended circulation
About 200 relatives of detained demonstrators took part in a protest outside Tehran's Revolutionary Tribunal calling for their release, AFP reported
EU foreign ministers expressed "serious concern" and called for an inquiry into the conduct of the election.
On Sunday Mr Mousavi's website carried a statement saying he had formally called on Iran's Guardian Council ,which must certify the counting, to annul the election.
He added: "I urge you, Iranian nation, to continue your nationwide protests in a peaceful and legal way."
Students chant 'resign, resign' at a secret rally
Another defeated candidate, Mohsen Rezaie, also officially contested the results. The Guardian Council said it would rule on both complaints within 10 days.
Late on Sunday Mr Mousavi met Iran's Supreme Leader, Ayatollah Ali Khamenei, to pursue his case against the results of Friday's election.
Mr Khamanei's office said he had told the candidate that "complaints should be followed through legal channels" and urged Mr Mousavi to "follow the issue calmly".
More than 100 opposition figures arrested, including the brother of ex-reformist President Khatami.
Local and international phone and text message services interrupted
Social networking and newspaper websites blocked
BBC says "heavy electronic jamming" from inside Iran disrupts its Persian TV service
International journalists arrested and asked to leave
Iranian newspapers do not carry reports of the violence
Source: Various reports
Sunday saw clashes between the police and anti-Ahmadinejad protesters in several parts of Tehran for a second day. Police hit protesters with batons and charged them on motorbikes.
Mousavi supporters cried "death to the dictator" into the evening. Scores of people are reported to have been arrested.
Mr Ahmadinejad dismissed the unrest as "passions after a soccer match".
Foreign powers have expressed concern about the election.
On Sunday, Vice-President Joe Biden said: "It sure looks like the way they're suppressing speech, the way they're suppressing crowds, the way in which people are being treated, that there's some real doubt."
German Foreign Minister Frank-Walter Steinmeier said he had summoned the Iranian ambassador to explain the election.
"There are a lot of reports about electoral fraud," he said.
French Foreign Minister Bernard Kouchner said: "I am sorry that instead of openness there has been a somewhat brutal reaction."
But Mr Ahmadinejad was congratulated by the presidents of Iraq, Afghanistan and Venezuela.
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