"We've used all the possible avenues to find with Russia a mutually acceptable solution", said EU Trade Commissioner Karel De Gucht.
The EU says the fee, introduced days after Russia joined the WTO last year, is discriminatory because domestic vehicles are exempted as are those imported from Kazakhstan and Belarus which are in a customs union with Moscow.
The request for WTO settlement comes after the EU launched proceedings at the Geneva-based trade body last July in view of a friendly settlement.
In Moscow, Maxim Medvedkov, Trade Director at the Ministry of Economic Development, said the decision "is as unexpected as inexplicable".
He said the move came the day after the Duma had agreed legislation in an initial vote to harmonise the levy between Russian producers and importers.
"The European Commission is refusing to seek a solution acceptable to both sides," he added.
Levied on cars, trucks, buses and other motor vehicles, it ranges from about 420 to 2,700 euros for a "new" vehicle and from 2,600 to 17,200 euros for a vehicle older than three years.
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