They seized the regional government's headquarters and prosecutor's office before opening fire with automatic weapons at the main police station.
Interim President Olexander Turchynov criticised local police for their "inaction" and "criminal treachery".
The US accused Russia of seeking to "change the security landscape" of Eastern and Central Europe.
In a speech at the Atlantic Council in Washington, Secretary of State John Kerry told the Kremlin to "leave Ukraine in peace" and warned: "Nato territory is inviolable we will defend every single inch of it."
In other developments on Tuesday:
A conference in London heard allegations that Ukraine's ousted President Viktor Yanukovych and his associates may have stolen assets worth tens of billions of dollars
Ukraine's Interior Minister Arsen Avakov told BBC Russian that voting in next month's presidential election may not be able to take place in all regions because of the unrest
'No control'
Moscow has said it has no intention of invading eastern Ukraine, where pro-Russia activists have seized government buildings in more than a dozen towns and cities.
Until now, only the local office of the State Security Service (SBU) in Luhansk, a city of 465,000 people less than 30km (20 miles) from the Russian border, had been targeted.
But on Tuesday afternoon, hundreds of people shouting "Russia, Russia" gathered outside the headquarters of the regional government to demand a referendum on greater autonomy.
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