US President Barack Obama has urged Russian president Vladimir Putin to make pro-Russian separatists in eastern Ukraine stand down, after violent clashes there and Russian troops reportedly massing on the border.
The phone call between the two leaders followed pro-Russian activists occupying buildings in eastern towns in the country.
Mr Putin said reports Russia was interfering in this were "unreliable", the BBC reported.
The phone call followed a meeting of EU foreign ministers in Luxembourg on Monday, where they called upon the separatists to stop "destabilising" Ukraine told Russia to pull back its troops from its border with the country.
UK Foreign Minister William Hague said there that Russian claims it was not interfering lacked "a shred of credibility".
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 said: "The costs Russia already has incurred will increase if those actions persist".
Speaking after Monday's meeting in Luxembourg, Baroness Ashton told reporters: "We condemn unreservedly the actions by armed individuals in the cities of eastern Ukraine.
"These attempts at destabilising Ukraine must stop. We strongly support the unity, sovereignty and territorial integrity of Ukraine.
"We call upon Russia to do so as well and pull back its troops from the Ukrainian border.
"Any further actions aimed at destabilising Ukraine have to stop.
At the weekend, Ukranian interim president Oleksandr Turchynov made a televised address in which he told the nation that it was at war with Russia, after pro-Russian forces took over government buildings and a Ukrainian security officer was killed by militia.