US Secretary of State John Kerry has warned Russia that NATO territory was inviolable and the alliance would defend "every piece" of it.
"Today Russia seeks to change the security landscape of Eastern and Central Europe," Mr Kerry said in a speech in Washington.
"We have to make it absolutely clear to the Kremlin that NATO territory is inviolable we will defend every single piece of it."
Mr Kerry described the events in Ukraine as a "wake-up call" to NATO allies.
He said the crisis puts "our entire model of global leadership" at stake.
Mr Kerry was speaking at an event about US-Europe ties at the Atlantic Council think-tank and told those gathered that what "Russia's actions in Ukraine tell us is that today President Putin is playing by a different set of rules".
Earlier today, Pro-Russian separatists took control of the regional prosecutor's office and television centre in the eastern city of Luhansk.
The city is in the heart of an area that Russian President Vladimir Putin described earlier this month as "New Russia", reviving a term from when the tsars conquered it in the 18th and 19th centuries.
Most people who live in the area now identify themselves as Ukrainians but speak Russian as a first language.
Witnesses reported separatists inside the prosecutor's office and gunmen guarding the entrance of the television centre.
The gunmen said they were in control of the building and Interfax news agency said protesters had burned the Ukrainian flag.
A Reuters witness said the gunmen opened fire with automatic weapons and threw stun grenades at the local police headquarters.
Around 20 gunmen shot at the building, trying to force police to surrender their weapons.
Police later abandoned the building and left the scene in buses that had been sent to collect them as an angry crowd shouted at them to "go home".
A group of separatists also seized the government headquarters in Luhansk.
The country's acting president Oleksander Turchynov has criticised the "inaction" and "treachery" of police in east Ukraine.
He demanded the dismissal of police commanders in Luhansk and Donetsk.
In a statement, Mr Turchynov said that events in east Ukraine had "shown the inaction, powerlessness and in some cases criminal treachery of the law enforcement authorities".
An aide to interim Interior Minister Arsen Avakov said: "The regional leadership does not control its police force.
"The local police did nothing."
OSCE calls for immediate release of monitors
The head of the Organisation for Security and Cooperation in Europe met Ukrainian authorities and the US ambassador in Kiev today, as intense, behind-the-scenes negotiations continued to free seven of the group's staff held by pro-Russian militants.
Secretary General Lamberto Zannier met Ukrainian Foreign Minister Andriy Deshchytsia, who demanded the "immediate release" of the seven Europeans detained in Slaviansk.
Mr Zannier declined to give an update on the fate of the group - including four Germans, one Dane, one Czech and one Pole - telling reporters only that "we are this moment discussing what to do".
Following talks with Mr Zannier and senior Ukrainian officials, the US ambassador to Kiev, Geoffrey Pyatt, demanded the OSCE staff be released "without conditions".
Rebels holding the group have previously described them as "prisoners of war" and said they will only be freed in exchange for the release of militants in custody.
They are also holding four Ukrainians, whose fate is also unclear.
Eight of the European monitors appeared before the cameras on Sunday and one of them said they were in good health.
One of the men, a Swede, was later freed on medical grounds as he has diabetes.
The self-proclaimed mayor of Slaviansk, Vyacheslav Ponomaryov, offered no comment to reporters waiting outside the town hall, where the captives are being held.
The OSCE is in Ukraine to assess progress towards an 17 April 17 accord struck in Geneva that was supposed to defuse tensions.
Part of that accord was that pro-Russian militants should leave occupied buildings but this has not happened, with the West accusing Moscow of failing to exert its influence over them.
EU imposes new sanctions on Russians
The European Union has imposed asset freezes and travel bans on 15 Russians, including a deputy prime minister and a deputy chairman of the State Duma, over Russia's actions in Ukraine.
Others on the list released today include the chief of staff of Russia's armed forces and separatist leaders.
However, the list did not include the heads of Russian energy giants such as Rosneft's Igor Sechin, who was included on a new US sanctions list yesterday.
The decision brings to 48 the number of people that the EU has put under sanctions for actions it says have undermined Ukraine's territorial integrity.
Russia's Foreign Ministry has criticised the new sanctions, saying the EU is under the thumb of the US.
It said the EU should be ashamed and that the sanctions against will not help stabilise Ukraine.
Russia annexed the Crimea region after Ukraine's pro-Russia president was ousted in February by protesters demanding closer links with Europe.
Ukraine and the West accuse Russia of stirring up a separatist campaign in the east, a charge the government in Moscow denies.
The EU decision coincided with an earlier White House announcement that the US was imposing sanctions on seven Russians and 17 companies linked to Russian President Vladimir Putin.
The US has been much more aggressive in the penalties it has imposed on Russia than the EU has been, which depends heavily on Russia for energy and has close trading links.
The EU has so far only put sanctions on individuals, not companies.
President Putin said tonight that Russia saw no need for counter sanctions against the West, but could reconsider the participation of Western companies in its economy, including energy projects, if sanctions continued.
"We would very much wish not to resort to any measures in response," he told reporters after meeting leaders of Belarus and Kazakhstan.
"But if something like that continues, we will of course have to think about who is working in the key sectors of the Russian economy, including the energy sector, and how."