US Secretary of State John Kerry told the Munich Security Conference that Russia must stop targeting moderate rebels in Syria and pull its troops out of Ukraine.
"To date, the vast majority of Russia's attacks (in Syria) have been against legitimate opposition groups," Kerry told the audience.
"To adhere to the agreement it made, Russia's targeting must change," he said, referring to the international deal forged yesterday, in which foreign ministers agreed to seek a "cessation of hostilities" in Syria within a week.
He spoke shortly after Russian Prime Minister Dmitry Medvedev said the world had "slid into a new period of Cold War."
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"Almost every day we are accused of making new horrible threats either against NATO as a whole, against Europe or against the US or other countries," Medvedev said.
A panel of eastern European leaders were eager to add to the criticism of Russia's assertive foreign policy.
He addressed Russia's president, who was not present, saying: "Mr (Vladimir) Putin, this is not a civil war in Ukraine, this is your aggression. This is not a civil war in Crimea, this is your soldiers who occupied my country."
Kerry emphasised that sanctions on Russia would remain in place until it implements all aspects of the Ukraine peace agreement reached in Belarus' capital Minsk last year.
"Russia has a simple choice: fully implement Minsk or continue to face economically damaging sanctions," he said.
"Isolationism, intolerance, disrespect of human rights, religious fanatics, homophobia -- this alternative Europe has a leader. His name is Mr Putin."
By contrast, Medvedev had earlier criticised the expansion of NATO and EU influence deep into formerly Soviet-ruled eastern Europe, which Russia still sees as its sphere of influence.
"European politicians thought that creating a so-called belt of friends at Europe's side, on the outskirts of the EU, could be a guarantee of security, and what's the result?" he said. "Not a belt of friends but a belt of exclusion."