The Islamic republic does not recognize dual nationality, and Tehran has bluntly told Washington the affair is none of its business.
A third US-Iranian, who is not jailed but has had her passport confiscated, has been accused of working for a "counter-revolutionary" radio station, Iran’s ISNA news agency reported Wednesday.
A fourth US-Iranian is reportedly under arrest, and a US former FBI agent has gone missing in Iran since early March.
"The United States is dismayed by the Iranian government’s decision to harass, and in some cases imprison, a number of American citizens on groundless charges," said State Department spokesman Sean McCormack.
"These academics, scholars and journalists, many of them in Iran to visit relatives, pose no threat to the Iranian government. Indeed, many of them have served for years as bridges between our two cultures, working to enhance understanding between our countries."
US officials "urge the Iranian authorities to release immediately the Americans in their custody, as well as return the passports of others being kept in Iran against their will."
Washington also called on Tehran "to cooperate in the case of American citizen Robert Levinson," McCormack said, referring to the former FBI agent missing in Iran since March 8.
On Wednesday Iran accused scholar Haleh Esfandiari and urban planner Kian Tajbakhsh of espionage. Hassan Hadad, the Tehran deputy prosecutor in charge of security issues, said the pair "admitted carrying out activities but said that their intention was to help Iran. Individuals have been identified in Tehran who are linked to this affair."
The third US-Iranian, Parnaz Azima, who is still at liberty, has been "accused of collaboration with Radio Farda, a counter-revolutionary radio," Hadad said.
Esfandiari heads the Middle East program at the Washington-based Woodrow Wilson International Centre for Scholars. She was arrested in March after returning to Iran to visit her 93-year-old mother.
Tajbakhsh has worked for the Open Society Institute of US billionaire George Soros, which Iran accuses of seeking to encourage a "Velvet Revolution."
US rights groups and hardline media in Iran have reported the arrest of US-Iranian businessman Ali Shakeri, but Hadad said that Tehran prosecutors were not involved.
Shakeri works for a private conflict-resolution group called the Center for Citizen Peacebuilding, while Azima works for Radio Free Europe’s Persian arm, Radio Farda.