"We have American ambiguity that is very damaging for us," Bassma Kodmani, member of the main opposition High Negotiations Committee which attended last month's peace talks in Geneva.
The committee has rejected Assad's demand for any transitional government to include his regime as Syria struggles to emerge from five years of civil war.
The White House last week indicated Assad should not feature in a transitional unity administration, White House spokesman Josh Earnest dubbing his participation a "non-starter" for Washington.
But Kodmani said the committee wanted confirmation of that stance two days after high level US-Russian discussions on ways of strengthening a fragile ceasefire.
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"We don't know what the United States are discussing with Moscow," Assad's long-time ally, said Kodmani.
"We are awaiting confirmation that the USA are maintaining their position to refuse to rehabilitate Assad," she told French media.
Kodmani stressed "Assad's departure must be negotiated. The end of the regime must be a controlled, not a chaotic, transition operation."
But she warned that if Moscow "continues to think Assad should continue to govern then we shall not have a solution in Syria. He cannot remain in power.
"The opposition's position is clear -- negotiation will occur while Assad is still in power, but the transition cannot happen with him."
UN special envoy Staffan de Mistura wants fresh peace talks to start next week in Geneva on ending a conflict that has killed more than 270,000 dead with a transitional government being formed in six months to draft a new constitution ahead of presidential elections in 18 months time.