Ex-deputy prime minister Boris Nemtsov, television host Ksenia Sobchak and liberal activist Ilya Yashin were interrogated again for their role in organizing recent rallies, including a May 6 protest that ended in violent clashes with police.
Putin has taken a tougher approach toward the opposition since he began his third term as president in May.
On Monday, police searched the properties of all three leaders and others. A day later, police brought them in for questioning just an hour before the massive anti-Putin protest in Moscow.
Sobchak tweeted that she was brought into a "strange, damp room" on Friday where she was questioned about the $1.7 million in cash found in her apartment. The wealthy TV host has said previously that she does not trust Russian banks.
Both Sobchak and Yashin were released after several hours of interrogation, but both said it was clear that the investigations were going to continue.
"I still don't understand what this investigation is leading toward," Sobchak said afterward. "The questions are very strange."
Sobchak tweeted that, before her passport with an American visa was returned, the investigator asked her if she knew anything regarding the 1963 assassination of President John F. Kennedy. Her money was not returned.
Until recently, Sobchak had been considered untouchable because of Putin's enduring loyalty to her late father, who as mayor of St. Petersburg in the early 1990s gave Putin his first government job.
Yashin said he could only guess what the proceedings would entail, because Russia's powerful Investigative Committee would not discuss the "protocol" for its probe.
"Throwing out a mention of my possible arrest was clearly meant to provoke me to leave the country," Yashin tweeted after his own release. "I don't intend to run away."
Investigative Committee spokesman Vladimir Markin said the investigation into the Russian opposition leaders was ongoing and that his agency was still reviewing all the documents seized in its search.
"The legal status of some of these people may change from witnesses to suspects," he said.
Investigations also continue for seven other opposition leaders and activists, including Left Front leader Sergei Udaltsov and anti-corruption blogger Alexei Navalny.