Around 40 gunmen in balaclavas and military fatigues fired shots above a convoy of unarmed observers from the Organization for Security and Co-operation in Europe (OSCE) on Saturday, preventing them from entering Ukraine's Crimean peninsula.
It was the third consecutive day observers were forced to turn back, but the first time shots were fired.
A spokeswoman for the OSCE said no one was hurt.
OSCE observers are in Ukraine on the request of the new interim government to monitor the crisis. However Russia claims they have failed to obtain an official invitation from Crimean authorities.
In a further escalation of tensions on Saturday, Ukraine's border guards reported that one of their light patrol plane came under fire from "extremists" in the same area but was unharmed.
Russia tightens grip on Crimea
Tensions between the Ukraine and Moscow have surged since an estimated 30,000 pro-Russian soldiers took control of the Crimean peninsula last week.
Concern increased two days ago when Crimea's parliament announced plans to hold a referendum on March 16 on joining the Russian Federation.
Putin justified Russian intervention earlier in the week, saying he hoped to protect the Russian-speaking majority in the region following last month's uprising in Kyiv that led to the ouster of President Viktor Yanukovych. He also denied that Russian armed forces were active in Crimea and instead called them "local self-defence forces."
Nevertheless, the news agency Associated Press reported on Saturday that dozens of military trucks, believed to be Russian, were again seen transporting heavily armed soldiers in Crimea.
The crisis has resulted in the worst face-off between the West and Moscow since the Cold War.
West scrambles for response
The White House said during a telephone conference on Saturday US President Barack Obama and the heads of Britain, France, Italy, Lithuania, Latvia and Estonia called on Russia to withdraw its military forces and to allow international observers into Crimea.
"The leaders reiterated their grave concern over Russia's clear violation of international law and reaffirmed their support for Ukraine's sovereignty and territorial integrity."
The Baltic countries, who are NATO members with strong economic ties to Russia and their own Russian minorities, have expressed anxiety over Moscow's actions.
The French presidency issued its own statement saying President Hollande and Obama had discussed new measures against Russia.
Separately, US Secretary of State John Kerry warned his Russian counterpart that room for negotiation was quickly closing.
In a phone conversation with Russia's Foreign Minister Sergei Lavrov, Kerry said any attempt to annex Crimea to Russia and continued military escalation would "close any available space for diplomacy."
While Russia's foreign minister has ruled out direct talks with Ukraine's interim government, which Russia does not recognize, Russia's deputy foreign minister did meet the Ukrainian ambassador to Moscow in Russian capital on Saturday.
"On the 8th of March, a meeting took place between Russian Deputy Foreign Minister Grigory Karasin and Ukraine's Ambassador to Russia Volodymyr Yelchenko during which, in an open atmosphere, questions of Russia-Ukrainian relations were discussed," Russia's foreign ministry said in a statement.
No further information was given on the extent to which the Crimea crisis was discussed.
The talks came hours after Lavrov advocated what he called "objective" talks with foreign powers.
'Objective' talks needed, says Lavrov
"We are open to an honest, equal and objective dialogue with our foreign partners to find a way to help all of Ukraine come out of the crisis," Russia's top diplomat said at a televised news conference in Moscow.
Lavrov warned, however, that dialogue would only go ahead if there were no attempt to "display Russia as a party to the conflict."
"This crisis was not created by us. All the more, it was created in defiance of our repeated and longstanding warnings," Lavrov said.
"The crisis has been created artificially, out of geo-political motives," he added, challenging Western accusations that Russia was responsible for the onset of the crisis.
ccp/se, ipj (AFP, dpa, Reuters, AP)