A second round of UN-led peace talks, dubbed Geneva II, broke down in acrimony earlier this month, threatening international attempts to mediate an end to Syria's vicious civil war.
International envoys are trying to get the process back on track, but Washington accused Bashar al-Assad's regime of using strong-arm tactics to intimidate opposition negotiators.
"We call on the regime to immediately and unconditionally release all those unfairly arrested," State Department spokeswoman Jen Psaki said, expressing Washington's "outrage."
"The opposition delegation must be permitted to safely and securely work towards the political transition," she said.
The impetus behind the Geneva dialogue came from a joint US-Russian diplomatic initiative, but the rival powers remain themselves deeply divided over the crisis in Syria.
US Secretary of State John Kerry is due to meet his Russian counterpart Sergei Lavrov in Rome next week for their first face-to-face meeting since the last round of talks failed.
But he was critical of Moscow's stance, accusing the Kremlin of increasing its supply of weapons to Assad's forces.
"Frankly, Russia is increasing its assistance to Assad," Kerry said.
"I do not find that constructive in the effort to try to get him to change his mind and be able to come to a decision that he needs to negotiate in good faith."
What Assad "is doing is outrageous, unconscionable, unacceptable, disgraceful, craven, it's horrendous. And we all know that. Everybody knows that," Kerry said, in an interview with MSNBC television.
One tangible result of the US-Russian diplomatic push was last year's agreement by Assad to hand over his chemical weapons arsenal for destruction by international monitors.
A fourth consigned of such arms -- in this case mustard gas -- was shipped put of the Syrian port of Latakia on Wednesday.
But the disarmament process is behind schedule and meanwhile fighting continues in what is now a many-sided battle between opposition rebels, Islamist guerrillas and loyalist forces.
Rebel ranks have been bolstered by foreign Jihadi fighters, and the regime has the support of the Iranian-backed Lebanese Hezbollah militia, fuelling fears that the conflict may spread.
The presence of Al-Qaeda linked extremists among the opposition forces has raised concerns in the West, but Washington places the bulk of the blame for lack of progress in talks on Assad.
"Unfortunately, these arrests and attempts to silence dissent are not new behavior for the Assad regime," Psaki said.
"This regime continues to brutalize the Syrian people through aerial bombardment and other indiscriminate attacks that maim and kill civilians by the thousands," she said.
"Political and arbitrary imprisonment, and the systematic torture and death of tens of thousands of people without access to due process, are but a few of the regime's documented human rights violations."
Among those held was Mahmoud Sabra, the brother of Geneva opposition delegation member, Mohammed Sabra.
Psaki said that through such actions the Syrian regime was "not only defying the international community but also seeking to suppress the legitimate aspirations of the Syrian people."