The heads of Russia's two houses of parliament said they would respect a decision by lawmakers in Ukraine's majority-Russian peninsula to renounce ties with Kiev and stage a March 16 referendum on switching over to Kremlin rule.
"Should the people of Crimea decide to join Russia in a referendum, we... Will unquestionably back this choice," said speaker of the upper house Valentina Matviyenko.
"We will respect the historic choice of the people of Crimea," said her lower house counterpart Sergei Naryshkin.
It marked the leaders' second lengthy phone call in five days and both sides described it as tough.
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The White House said Obama "emphasised that Russia's actions are in violation of Ukraine's sovereignty and territorial integrity, which has led us to take several steps in response, in coordination with our European partners."
The Kremlin for its part said Putin tried to calm tensions by stressing that US-Russian relations "should not be sacrificed due to disagreements over individual -- albeit extremely significant -- international problems."
Yet with Russian forces in effective control of Crimea -- a predominantly ethnic Russian peninsula roughly the size of Belgium and the base of the Kremlin's Black Sea Fleet -- the threat of Ukraine's division seemed more real than at any point since Putin won parliamentary approval to use force against his western neighbour.
Western allies have been grappling with a response to Putin's seeming ambition to recreate vestiges of the Russian empire without regard to the damage this does to Moscow's foreign relations or instability it creates.
Putin has previously denounced the interim leaders' rise to power as an "unconstitutional coup".
The tensions in Ukraine intensified still further when the city council of Sevastopol that houses the Kremlin's Black Sea Fleet also resolved to become "a subject of the Russian Federation" with immediate effect.