The expulsion follows Czech media reports on Monday that two Russians had been ordered out of Prague, including a deputy military attache. Prague has previously complained about an increase in Russian spying, linking it to the U.S. plans.
Formerly part of the Soviet bloc, the Czech Republic is now a member of NATO and the European Union. Its relations with Moscow have cooled over its interest in hosting a U.S. radar base and its support for Georgia during last August's war.
"This unfriendly act by the Czech side, which declared two of our diplomats 'persona non grata', could not be left without a response," the Russian official told Interfax. "Two Czech embassy workers in Moscow were told to leave Russia."
"It is surprising that this information became public knowledge despite the fact that the Czech side itself proposed not to allow any leaks," the Russian official told Interfax.
The comment suggests that Russis had anticipated the expulsions, but had not expected them to become public.
Russia's Foreign Ministry routinely uses Russian news agencies as its main channel to distribute information.
Russia has also been involved in the reciprocal expulsion of diplomats in recent months with both Ukraine and NATO, and the latest row fits into a pattern pointing to Moscow's poor relations with many European countries, said an analyst.
"How far-sighted is this Russian policy of responding in kind to anything regarded as unfriendly? The fact is that as a result of Putin's policies, it doesn't have a single reliable ally," said Masha Lipman of the Moscow Carnegie Centre.
Russia can be a difficult, tough partner for many countries who in turn view it with suspicion and hostility, she said.
"In Central Europe, Russia has achieved its goal and the U.S. is no longer as anxious to install the missile shield as it was under President Bush. But apparently it wants guarantees, and that will never happen, it's something Russia won't get."
The Czech expulsions were first reported by the website www.idnes.cz on Monday.
When asked about the report, Russian Foreign Minister Sergei Lavrov said it was "another provocation", but did not make clear if he was referring to the report or the expulsions themselves.
Russia's Foreign Ministry declined repeated requests by Reuters for comment, including a written question on Monday.
The Czech Foreign Ministry and the Czech embassy in Moscow both also declined comment.
(Additional reporting by Dmitry Solovyov and by Michael Winfrey in Prague)
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