The row is the biggest between Prague and Moscow since the end of decades of Soviet domination of eastern Europe in 1989.
It also adds to growing tensions between Russia and the West in general, raised in part by Russia's military build-up on its Western borders and in Crimea, which Moscow annexed from Ukraine in 2014, after a surge in fighting between government and pro-Russian forces in Ukraine's east.
Russia said Prague's accusations were absurd as it had previously blamed the blast at Vrbetice, 300 km (210 miles) east of the capital, on the depot's owners.
It called the expulsions "the continuation of a series of anti-Russian actions undertaken by the Czech Republic in recent years", accusing Prague of "striving to please the United States against the backdrop of recent U.S. sanctions against Russia".
Jan Hamacek, the Czech interior and acting foreign minister, said investigators believed the blast had been intended to occur in an arms shipment after it left the depot, probably headed for Bulgaria.
Czech police said they were looking for two men who travelled to the Czech Republic days before the blast under the names Alexander Petrov and Ruslan Boshirov.
Those names were the aliases used by the two Russian GRU military intelligence officers wanted by Britain for the poisoning of former Russian spy Sergei Skripal and his daughter with the Soviet-era nerve agent Novichok in the English city of Salisbury in 2018. The Skripals survived, but a member of the public died.
The Kremlin denied involvement in that incident, and the attackers remain at large.
"Police knew about the two people from the beginning," Hamacek said, "but only found out when the Salisbury attack happened that they are members of the GRU, that Unit 29155."
Hamacek said Prague would ask Moscow for assistance in questioning them, but did not expect it to cooperate.
The Czech investigative weekly Respekt reported on Saturday that according to police investigators, the arms shipment was destined for a Bulgarian trader believed to be supplying Ukraine at a time when Russian-backed separatists were fighting government forces in eastern Ukraine.
Respekt and Czech public radio named a Bulgarian arms dealer, a man whom Bulgarian prosecutors said Russian agents had tried and failed to kill in 2015.
The news website Seznamzpravy.cz said the arms shipment may also have been destined for Syrian rebels.
"DANGEROUS AND MALIGN"
British Foreign Secretary Dominic Raab tweeted that the Czechs "have exposed the lengths that the GRU will go to in their attempts to conduct dangerous and malign operations".
A NATO official said the alliance would support the Czech Republic as it investigated Russia's "malign activities", which were part of a pattern of "dangerous behaviour".
"Those responsible must be brought to justice," added the official, who declined to be named.
Washington also offered Prague its support.
The United States imposed sanctions against Russia on Thursday for interfering in last year's U.S. election, cyber hacking, bullying Ukraine and other actions, prompting Moscow to retaliate.
On Sunday, U.S. national security adviser Jake Sullivan said Washington had told Moscow "there will be consequences" if Alexei Navalny, the opposition figurehead who almost died last year after being given a toxin that Western experts say was Novichok, dies in prison, where he is on hunger strike.
The 2014 incident has resurfaced at an awkward time for Prague and Moscow.
The Czech Republic is planning to put the construction of a new nuclear power plant at its Dukovany complex out to tender.
Security services have demanded that Russia's Rosatom be excluded as a security risk, while President Milos Zeman and other senior officials have been putting Russia's case.
In a text message, Industry Minister Karel Havlicek, who was previously in favour of including Russia, told Reuters: "The probability that Rosatom will participate in the expansion of Dukovany is very low."
(Additional reporting by Jan Lopatka in Prague, Philip Blenkinsop in Brussels, Elizabeth Piper in London and Andrew Osborn in Moscow Editing by Kevin Liffey and Mark Heinrich) ((firstname.lastname@example.org; +420 234 721 614))