In the video, a man resembling Igor Bezler, a Russian separatist leader in the eastern Ukrainian town of Horlivka, demanded that Kiev release a separatist prisoner in its custody, or he would execute more Ukrainian hostages.
The man, wearing military fatigues and armed with a Kalashnikov rifle, is seen standing in a dark room where two blindfolded men are at the far side facing a wall.
“If my man is not released in an hour, another two will be shot; in another hour and a half a third two,” he says, addressing Kiev’s leadership.
The two alleged hostages in the video are then seen falling to the ground to the sound of machine gun shots.
The footage – if confirmed – would be among the most graphic of the worsening violence in the east, where Kiev and Moscow have accused each other of using “terrorist” tactics.
Since the separatist revolt began in March, dozens of people have disappeared or been taken into captivity in rebel-controlled towns in the region.
Some were later found dead.
The authenticity of the video, first reported on by local media in Horlivka, could not be independently verified.
However, a representative of the separatist authorities that took charge in Ukraine’s eastern Donetsk province after a referendum last month said the shooting shown in the video was authentic.
“I can confirm that this happened,” Alexander Khryakov, a leader of the Donetsk People’s Republic, said.
After the video spread on Ukrainian news sites on Thursday, Oleksandr Turchynov, the interim Ukrainian president, accused Russia of supporting “bloody terror” in eastern Ukraine.
Kiev and its western allies have increasingly accused Moscow in recent days of funnelling arms and men to the eastern revolt.
“Responsibility for the bloody crimes of Russian Federation Citizen Bezler also hangs on the political leadership of Russia, which continues to arm and support bloody terror in eastern Ukraine,” Mr Turchynov said in a statement on the presidential website.
An aide to a senior security official in Kiev confirmed that Ukrainian servicemen had been taken hostage by the rebels, but would not discuss the number in captivity.
The man speaking in the video makes reference to Ukrainian army officers, SBU security officials, and policemen in captivity.
In discussing the video, Mr Khryakov blamed Kiev for breaking the terms of an agreed prisoner swap.
He said a female separatist prisoner in Ukrainian custody had been scheduled for release under an exchange, but was kept under arrest on Mr Turchynov’s orders.
“Our army kept up its side of the bargain,” Mr Khryakov said.
“They betrayed their own.”