Ahmad al-Asrawi and Munir al-Bitar, two members of the National Coordination Committee for Democratic Change, 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.
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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.
The committee is set to choose at least part of an opposition delegation for peace talks with the government next month.
"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 the pair was taken "to an unknown location".
"Those who want a political solution would not do this," he said.
In a statement published online, the NCCDC said 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 arrest comes less than a week after Syria's army claimed responsibility for the killing of rebel Chief Zahran Alloush.
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Alloush was the head of Jaish al-Islam, the most powerful rebel faction in the Damascus province.
Jaish al-Islam, too, had taken part in the opposition meeting in Riyadh.
Saudi Foreign Minister Adel al-Jubeir on Tuesday blamed Alloush's death on Russia, saying the killing does "not serve the peace process and (efforts) to achieve a political solution in Syria."
Syria's government has regularly referred to Jaish al-Islam and to Alloush as "terrorists," but has said it is ready to participate in new peace talks.
On Wednesday, Prime Minister Wael al-Halqi said Syria was "ready to take part in Geneva talks next month... without any international interference."
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