The Ukraine city of Sevastopol has declared itself a subject of the Russian Federation, hours after the semi-autonomous parliament of Crimea voted to do the same.
The city, on the Crimean peninsula and home to the Black Sea fleet of the Russian navy, made the announcement late on Thursday.
The US and EU earlier joined the Ukrainian government in condemning as "illegitimate" an earlier vote by the Crimean parliament to secede from Ukraine, and bring forward a planned referendum on the subject to March 16.
The move was condemned by Kiev as having no basis in law, while the US President Barack Obama said the referendum would violate the Ukrainian constitution and international law.
"Any discussion about the future of Ukraine must include the legitimate government of Ukraine," he said.
However, Rustam Temurgaliyev, the region's deputy prime minister, had earlier said the decree was effective immediately and that Russian soldiers were the only legitimate forces in Crimea.
"The Ukrainian armed forces have to choose: lay down their weapons, ... accept Russian citizenship and join the Russian military."
"If they do not agree, we are prepared to offer them safe passage ... to their Ukrainian homeland."
Ukraine's Prime Minister Arseniy Yatsenyuk said the vote was "illegitimate", while Oleksander Turchinov, the acting president, said Crimea MPs were working "under the barrel of a gun".
Obama also issued an executive order authorising sanctions against "individuals and entities responsible for activities undermining democratic processes or institutions in Ukraine.''
He said that Russia's involvement in Crimea constituted "an unusual and extraordinary threat to the national security and foreign policy of the United States".
Meanwhile, the European Union announced that it was to suspend talks with Russia on a wide-ranging economic pact and on a visa deal. It also threatened further sanctions if Russia did not quickly engage in talks to end the crisis.
After a meeting of EU ministers in Brussels, Donald Tusk, Poland's prime minister, said the EU members of G8 had also agreed to suspend preparations for a G8 summit in June in Sochi.
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Al Jazeera's Hoda Abdel-Hamid, reporting from Sevastopol, said there were serious questions about the legitimacy of the parliament, the prime minister, and the decree.
"The prime minister came to power arguably at gunpoint when the parliament was taken over," she said, referring to a takeover of the building by pro-Russian forces last week.
"The constitution also says parliament cannot take such a decision," she said, adding that Russia would also have to formally accept the decree.
About 11,000 pro-Russian troops are in control of the peninsula and have blocked all Ukrainian military bases that have not yet surrendered, according to the regional leader Sergei Aksyonov.
All or most of those troops are believed to be Russian, even though Moscow has repeatedly denied sending them.
Some of those troops on Thursday blocked military observers from the Organisation for Security and Co-operation in Europe from entering Crimea, the group said.
Diplomatic efforts to cool the crisis continued on Thursday.
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John Kerry, US Secretary of State, met his Russian counterpart Sergei Lavrov on the sidelines of a meeting in Rome, but little appeared to have been resolved.
"For the moment we cannot report to the international community that we are in agreement," Lavrov was quoted by Russian news agencies as saying.
Kerry "made clear the importance of the Russians talking directly to the Ukrainians", US diplomatic sources told journalists after the 40-minute meeting.
The US also announced that the guided-missile destroyer, the USS Truxtun , was heading to the Black Sea area, in a deployment planned before the Ukraine crisis began.
The Pentagon said it had sent six F-15 fighter jets to Lithuania to bolster patrols over the Baltic states.
According to reports in Poland, the US was also sending a squadron of F-16 fighter jets there for a training exercise.