Barak Obama. Photo:AFPUS President Barack Obama has urged his Russian counterpart Vladimir Putin to use his influence to make separatists in eastern Ukraine stand down, bbc reports.
The phone call between the two leaders came as pro-Russian activists continued to occupy buildings in eastern towns.
For his part, Mr Putin rejected accusations of Russian interference, calling the reports "unreliable".
Meanwhile, Ukraine's acting President, Olexander 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 armour being on the move near the flashpoint towns of Sloviansk and Kramatorsk.
Tanks and armoured personnel carriers could be seen parked 70km (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 destabilise the government of Ukraine," a statement said.
"The president emphasised 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".
It said Mr Putin had urged Mr 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".
UK Foreign Secretary William Hague said on Tuesday the situation in eastern Ukraine was "very dangerous". Speaking to the BBC, he commended the Ukrainian government for acting "very responsibly throughout this crisis".
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.
Rebels appeal to Putin
Mr Turchynov said on Tuesday the aim of the operation in Donetsk was to "protect Ukrainian citizens, to stop the terror, to stop the crime, to stop the attempts to tear our country apart".
Pro-Russian rebels have seized buildings in about 10 towns and cities across eastern provinces that form the heartland of Ukraine's heavy industry.
A four-way meeting on the crisis, involving the EU, Russia, the US and Ukraine is due to be held in Geneva on Thursday.
Russian Foreign Minister Sergei Lavrov warned on Tuesday that any use of force by the Ukrainian government in the east could undermine the talks.
"You can't send tanks against your own citizens and at the same time hold talks," he said.