The Czech government on Wednesday warned Moscow it might expel more Russian diplomats unless the 20 Czech nationals ejected from Russia were allowed to return to work within a day.
Moscow responded by saying the ultimatum was "unacceptable".
On Saturday, Prague expelled 18 Russian embassy staff in a row over Russia's alleged role in an explosion that killed two people in the Czech Republic in 2014. Moscow sent back the Czech diplomats in retaliation on Monday.
"The Russian Federation has until 1200 tomorrow (1000 GMT) to allow the return of all expelled diplomats back to the Czech embassy in Moscow," Jakub Kulhanek, the new Czech foreign minister, told reporters.
"If they cannot return, I will cut the number of Russian embassy staff in Prague so it would correspond to the current situation at the Czech embassy in Moscow," he added.
After summoning Russian ambassador Alexander Zmeyevski, Kulhanek said Moscow's retaliation had been "disproportionate and it in fact paralysed the embassy".
- 'Unacceptable tone' -
The Russian foreign ministry condemned the Czech position.
"We suggest Prague leave ultimatums for communication within NATO," said spokeswoman Maria Zakharova. "With Russia such a tone is unacceptable."
The Czech ambassador would be summoned on Thursday, she added.
Prague currently has five diplomats and 19 technical staff at the embassy in Moscow, far fewer than the Russian workforce in Prague.
"The expulsion of 18 Russian diplomats in turn did not jeopardise the functioning of the Russian embassy," said Kulhanek, who was only appointed as minister on Wednesday.
The EU backed the Czech Republic as its foreign policy chief Josep Borrell said the bloc stood "ready to support its further efforts to bring those responsible to justice".
"The EU condemns the disproportionate reaction and subsequent threats of Russian Federation towards the Czech Republic," Borrell said in a statement, vowing "the staunchest resolve" in addressing disruptive acts by Russian intelligence on EU soil.
- Reset relations? -
Czech officials, including Interior Minister Jan Hamacek, who was standing in as foreign minister until Kulhanek's appointment, said Tuesday that they might aim to reset relations with Russia -- and that this could involve the expulsion of all Russian diplomats in Prague.
Prague accused the Russian secret services Saturday of being behind an explosion at an ammunition depot near the eastern village of Vrbetice in 2014 that killed two Czech nationals.
Czech police are seeking two men in connection with that blast, as well as a second, non-fatal explosion nearby later the same year.
The British authorities have identified the same men as suspects in the 2018 poisoning of former Russian double agent Sergei Skripal in Salisbury, England.
The Czech attack allegedly targeted ammunition belonging to a Bulgarian arms dealer. Media reports in Bulgaria have suggested he had been exporting arms to Ukraine.
The explosion occurred the same year that Russia annexed the Crimean peninsula from Ukraine and a conflict broke out between Ukrainian forces and Russia-backed rebels in the east of the country.
The Czech government has already announced that Russia's atomic energy agency Rosatom would be excluded from a tender to build a new multi-billion-euro unit at a Czech nuclear plant.
Hamacek also said that Prague would no longer consider buying Russia's Sputnik V vaccine against Covid-19.