Russia has announced that 20 diplomats from the Czech Republic will be expelled after the Czechs expelled 18 Russian diplomats.
The Russian diplomats are suspected of being intelligence operatives involved in an explosion at a Czech arms depot in 2014 which killed two people.
Two Russians suspected of carrying out a nerve agent attack in the UK in 2018 are now also being linked to the blast.
EU foreign ministers are due to discuss the accusations.
Moscow has given the Czech diplomats a day to leave, while the Czech Republic has given the Russians 72 hours.
Russia's foreign ministry called the Czech decision "unprecedented" and a "hostile act".
"In their desire to please the United States against the background of recent US sanctions against Russia, Czech authorities in this respect even outdid their masters from across the pond," said a foreign ministry statement.