President Barack Obama has called President Vladimir Putin urging him to use his influence to make separatists in eastern Ukraine stand down.
The phone call between Barack Obama and Vladimir Putin came as pro-Russian activists continued to occupy buildings in eastern towns.
For his part, Vladimir Putin rejected accusations of Russian interference, calling the reports “unreliable”.
Meanwhile, Ukraine’s acting President, Oleksandr Turchynov, has announced the start of an “anti-terrorist operation”.
He told parliament it had begun in the “north of Donetsk Region” on Tuesday morning and was being conducted “stage by stage, in a responsible and weighed manner”.
The extent of the operation was unclear but unconfirmed reports on Russian media, quoting separatists, speak of Ukrainian armor being on the move near the flashpoint towns of Sloviansk and Kramatorsk.
Tanks and armored personnel carriers could be seen parked 44 miles from Sloviansk on Monday.
EU foreign ministers say they will expand a list of names targeted by sanctions.
Tension has been steadily rising since Russia annexed the Crimean peninsula, formerly part of Ukraine, last month.
The move, condemned as illegal by Kiev and the West, followed the ousting of Ukraine’s pro-Moscow President Viktor Yanukovych in February.
The White House said the “frank and direct” conversation between the two presidents was made at Russia’s request.
“The president expressed grave concern about Russian government support for the actions of armed, pro-Russian separatists who threaten to undermine and destabilize the government of Ukraine,” a White House statement said.
“The president emphasized that all irregular forces in the country need to lay down their arms, and he urged President Putin to use his influence with these armed, pro-Russian groups to convince them to depart the buildings they have seized.”
The statement also threatened Moscow with wider sanctions, saying “the costs Russia already has incurred will increase if those actions persist”.
The Kremlin said in a statement that recent unrest in Ukraine’s south-east was “the result of the unwillingness and inability of the leadership in Kiev to take into account the interests of Russia and the Russian-speaking population”.
The statement said Vladimir Putin had urged Barack Obama to “use the resources at the disposal of the American side” to help prevent any bloodshed.
It dismissed claims that Russia was interfering in Ukraine, saying the accusations were based on “questionable information”.
Thousands of Russian troops are reported to be deployed along the border between Ukraine and Russia. Kiev fears any crackdown on pro-Russian groups could trigger an invasion.
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