The arrest comes a week after his supporters took to the streets in their first demonstrations in more than a year.
"Last night security forces entered the house of Ali Karoubi and arrested him and his wife, Nafiseh Panahi," Sahamnews website said. Panahi was later released but there was no further news of Ali Karoubi, it said.
Sahamnews said there were concerns for Mehdi Karoubi himself after security forces raided his house on Monday night and confiscated documents and books.
"After last night's incident and despite many efforts there is no news of the fate of (Mehdi) Karoubi and his wife," the website said.
Mehdi Karoubi has been under virtual house arrest for more than a week after calling for supporters of his reformist Green movement to hold a rally on February 14, the first event of its kind since December 2009. More demonstrations were held on Sunday.
The new protests were inspired by events in Tunisia and Egypt which the Iranian government has itself applauded, perceiving the uprisings as part of an Islamic awakening.
The Iranian rallies were not authorised and were countered by a heavy security clampdown.
Along with Mirhossein Mousavi, the 73-year-old Karoubi heads the "Green" opposition movement which staged huge protests after the 2009 re-election of President Mahmoud Ahmadinejad which they said was rigged.
The government denies the charge and crushed those demonstrations, accusing the Green movement of trying to overthrow the Islamic system with the support of Iran's foreign enemies.
Government supporters have called for both opposition leaders to be tried and executed, but so far authorities have chosen to isolate rather than arrest them, wary of giving them greater publicity and angering their supporters.
Two people were shot dead at the February 14 rally in Tehran, deaths that government supporters blamed on "terrorist" elements among demonstrators but which the opposition movement said were evidence of state brutality.
Police in the southern city of Shiraz denied a third man had been killed during demonstrations there on Sunday.
Opposition websites said university student Hamed Nour-Mohammadi was thrown off a bridge. ISNA quoted a police official as saying there had been no protests in Shiraz and the student had died in a traffic accident.
Sahamnews said security forces were seeking another of Karoubi's sons, Hossein, who has been outspoken since his father's house arrest.
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