Alexander Bastrykin told Rossiiskaya Gazeta state daily that in the first Chechen war in the mid-1990s, Ukraine's premier "Arseny Yatsenyuk fought against Russian troops".
The claim was widely ridiculed in Ukraine, where Yatsenyuk often is portrayed in cartoons as the bossy but harmless Rabbit from the children's book "Winnie-the-Pooh".
Bastrykin said an "investigation had established" that Yatsenyuk, now 41, fought in hit squads called Argo and Viking.
The chief investigator said the units were led by Oleksandr Muzytchko -- better known as Sashko Bilyi -- the late coordinator of Right Sector far-right nationalist group in western Ukraine, who was shot dead by police last year.
The allegations caused a storm of sarcastic comments on Ukrainian social networks, with photoshopped images featuring Yatsenyuk, who is mocked for his alleged resemblance to the rabbit in the Soviet cartoon version of Winnie-the-Pooh.
Images show him sporting a long beard in full Rambo-style gear, surrounded by Islamists. One shows a rabbit with a grenade launcher with the caption: "He killed his first Russian at 16."
The interior minister Arsen Avakov also mocked the claim that Yatsenyuk, a balding pro-Western former foreign minister, used to fight alongside Islamist militants in Chechnya.
According to Yatsenyuk's official biography, in 1994 and 1995 he was working on privatisations as the head of a law firm.
Such allegations, published in a state newspaper, appeared to be aimed at reinforcing Moscow's line that Kiev is controlled by ultranationalist neo-Nazis.
Kremlin spokesman Dmitry Peskov backed the claim, telling Russian news agencies: "The Investigative Committee is working on it and such statements can never be groundless."
Right Sector played a key role in the frontlines of deadly protests that unseated pro-Moscow president Viktor Yanukovych last year and its well-equipped fighters have also fought pro-Russian separatists in the east.
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