Alexander Zakharchenko, who is prime minister of the self-declared Donetsk People’s Republic, told Russian TV many of the Russians were former servicepeople or current personnel on leave.
He was speaking as rebels threatened to take the key port of Mariupol, after opening a new front in the south-east.
Reports say they have captured the town of Novoazovsk and are advancing on the port.
Ukraine says Russian forces have crossed the border and are supporting the rebel attack, but Moscow has repeatedly denied arming or covertly supporting the rebels.
There are suspicions that Russia wants to divert Ukrainian forces from the besieged cities of Donetsk and Luhansk, further north.
Government forces have made significant advances against the separatists in recent weeks.
German Chancellor Angela Merkel has demanded an explanation from Russia’s President Vladimir Putin amid the reports of an incursion.
French President Francois Hollande also said it would be “intolerable” if Russian troops were in Ukraine.
Alexander Zakharchenko said Russian citizens were coming to Ukraine to fight because they felt it was their duty.
“There have been around 3,000-4,000 of them in our ranks,” he said, adding that the rebels’ struggle would have been much harder without them.
He insisted that any Russians fighting on the rebel side were doing so voluntarily.
“Many former high-ranking military officers have volunteered to join us. They are fighting with us, considering that to be their duty,” he said.
“There are also many in the current Russian military that prefer to spend their leave among us, brothers who are fighting for their freedom, rather than on a beach.”
Reports from journalists and military on the ground say that Novoazovsk has been captured by the rebels.
A Ukrainian company commander, Vladimir Shilov, told Ukrainian TV that he had heard from sources inside the town that it was blocked by tanks and no-one was allowed to leave. Local officials had already fled to Mariupol, he added.
A spokesman for the rebels told Interfax news agency that Novoazovsk was under their control and they would soon “liberate” Mariupol.
Reports on Twitter suggested the rebels were already advancing towards Mariupol, and rebels said they had captured checkpoints to the west of the city.
The port has until now been peaceful and cut off from rebel positions.
Rebels have been trying for weeks to break out of an area further north in the Donetsk region where they are almost encircled.
Analysts say the separatists could also be seeking a land link between Russia and Crimea, which also would give them control over the entire Sea of Azov.
Russia annexed the Black Sea peninsula of Crimea from Ukraine in March.
In a phone call with Vladimir, Angela Merkel said reports of a Russian military incursion into Ukrainian territory had to be cleared up, her spokesman said.
The US also expressed its “deep concern” at the latest developments.
“These incursions indicate a Russian-directed counter-offensive is likely under way in Donetsk and Lugansk [Luhansk],” state department spokeswoman Jen Psaki told reporters on Wednesday.
“An increasing number of Russian troops are intervening directly in fighting on Ukrainian territory,” the US ambassador to Ukraine, Geoffrey Pyatt, wrote on Twitter.
Ukrainian President Petro Poroshenko promised a roadmap for peace in the east on Tuesday after holding his first direct talks on the crisis since June with Vladimir Putin.
Vladimir Putin said Russia would assist any ceasefire talks, but that stopping the fighting was a matter for Ukraine alone.
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