Ahmad al-Asrawi and Munir al-Bitar, two members of the National Coordination Committee for Democratic Change (NCCDC), were stopped at Syria's border with Lebanon, said the body's secretary general, Yahya Aziz.
Both Asrawi and Bitar were headed to Saudi Arabia to join fellow members of the opposition's "supreme committee for negotiations," Aziz told AFP.
The "supreme committee" is a 33-member group formed earlier this month at a landmark meeting of Syria's armed and political opposition in Riyadh.
"Syrian authorities today arrested our two colleagues Ahmad al-Asrawi and Munir al-Bitar at the Syrian-Lebanese border point as they were heading to the supreme committee meeting in Riyadh," Aziz said.
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He said they were taken "to an unknown location".
"Those who want a political solution would not do this," he said.
The NCCDC said in a statement that the arrest contradicted "international efforts to reach a just political solution" to the conflict in Syria.
It demanded that Asrawi and Bitar be released and said Syrian authorities "were responsible for their safety".
"The regime and its allies Russia and Iran are not serious about the political process, (because) they are targeting members of the Riyadh conference that are committed to a political solution," the statement said.
The arrests come less than a week after Syria's army claimed responsibility for the killing of rebel chief Zahran Alloush.
Alloush was the head of Jaish al-Islam, the most powerful rebel faction in Damascus province.
Jaish al-Islam, too, had taken part in the opposition meeting in Riyadh.