The Czech government said it is expelling 18 Russian diplomats identified by local intelligence as secret agents of the Russian SVR and GRU services that are suspected of involvement in a 2014 explosion.
Czech police also said later they were seeking two Russians in connection with the blast, which killed two people, with passports used by the suspects in the attempted poisoning of former Russian double agent Sergei Skripal in 2018.
"Eighteen employees of the Russian embassy must leave our republic within 48 hours," Foreign Minister Jan Hamacek told reporters.
Prime Minister Andrej Babis said Czech authorities had "clear evidence" linking GRU officers from unit 29155 to the blast in a military ammunition warehouse near the eastern Czech village of Vrbetice.
He added that he had received the information on Friday, without explaining why the investigation had taken so long.
"The explosion led to huge material damage and posed a serious threat to the lives of many local people, but above all it killed two citizens," Mr Babis said.
The blast occurred on 16 October 2014 at a warehouse with 58 tonnes of ammunition. It was followed months later by another big blast at a nearby warehouse with 98 tonnes of ammunition.
The Czech organised crime squad (NCOZ) said it was looking for two men using Russian passports in relation to the explosions.
The passports bear the names of Alexander Petrov, born in 1979, and Ruslan Boshirov, born in 1978, and their holders are also wanted in Britain in connection with Mr Skripal's poisoning in Salisbury.
"The two men were present on Czech territory in... October 2014" when the Vrbetice blast occurred, the NCOZ said, adding that the two men also used Tajik and Moldovan identities.
Russia denied involvement in the poisoning but some 300 diplomats were sent home in subsequent tit-for-tat expulsions.
Russia has said it would respond to the "unprecedented" decision to expel a further 18 diplomats.
"We will take retaliatory measures that will force the authors of this provocation to understand their full responsibility for destroying the basis of normal relations between our countries," the Russian foreign ministry said in a statement.
Mr Babis said the expulsion of 18 diplomats had the full support of President Milos Zeman, a veteran left-winger who has fostered close ties with both Moscow and Beijing.
Mr Zeman has repeatedly spoken out against his country's Security Information Service (BIS), which has accused Russian intelligence services of orchestrating recent cyberattacks on the Czech foreign ministry and other targets.
Mr Hamacek said earlier this week he would travel to Moscow on Monday to negotiate potential supplies of the Russian-made Sputnik V vaccine, but he has cancelled the trip over the diplomatic spat.
Mr Hamacek, who is the interior minister and also an interim foreign minister after his predecessor was sacked earlier this week, said he was sorry the incident would "fundamentally damage Czech-Russian relations".
He said he had summoned Russian ambassador Alexander Zmeyevsky to convey the decision.
"We are in a situation similar to that in Britain following the attempted poisoning in Salisbury," he added.
On Thursday, Czech neighbour Poland said it had expelled three Russian diplomats for "carrying out activities to the detriment" of Poland.
