Leopoldo Lopez and Antonio Ledezma were both already under house arrest when they were picked up by the intelligence service known by its in acronym Sebin, the wife of Lopez and children of Ledezma said separately.
The two men are Venezuela's most high profile opposition leaders. Both had called for a boycott of Sunday's vote for a so-called and all-powerful constituent assembly tasked with rewriting the constitution.
Both of their families said they held President Nicolas Maduro, the driving force behind the vote, responsible for the leaders' lives.
"They just took Leopoldo away. We do not know where he is or where they are taking him," Lopez's wife Lilian Tintori said on Twitter.
The children of Ledezma - named Victor, Vanessa and Antonietta - also said on Twitter that the Sebin had taken away their father.
Opposition leaders and local media posted cell phone footage of Ledezma being taken away from his home forcibly.
Opposition lawmaker Freddy Guevara said the arrests were aimed at "frightening us and demoralising us."
