In a statement on Sunday, the US embassy in Ankara raised concerns over clashes in the southeast between protesters and police after 28 mayors were suspended mainly for suspected ties with the outlawed Kurdistan Workers' Party (PKK).
The embassy called for early local elections to replace the ousted mayors, who had been elected in 2014 polls, "in accordance with Turkish law."
The statement "is unacceptable, in particular for independent countries," Interior Minister Suleyman Soylu said, attributing the comments to US ambassador John Bass.
"That's what we expect because it is not the right approach," he said. "It is a test of sincerity. It is unacceptable."
Foreign Minister Mevlut Cavusoglu also conveyed Turkey's "concerns" over the embassy statement during a phone conversation with US Secretary of State John Kerry late Tuesday, foreign ministry sources said.
Speaking in the southern city Antalya Tuesday, Cavusoglu harshly criticised the embassy's statement, saying that the US ambassador was not "a governor" of Turkey.
Diplomats in the hugely sensitive post of American ambassador to Ankara often find themselves hit by brickbats from the government and Bass' predecessor Francis Ricciardone was often hit by similar criticism.
The row comes against the background of strained relations between the United States and Turkey, in the wake of the July 15 failed coup.
Ankara wants Washington to extradite Fethullah Gulen, who it accuses of masterminding the plot and is frustrated by American warnings that the process will take time.
The suspension of the 28 mayors was welcomed by President Recep Tayyip Erdogan as a decision long overdue.
But in a symbolic gesture, Soylu also announced he was opposed to the removal of a Kurdish-language signs from Diyadin municipality.
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