In Donetsk, Luhansk and Kharkiv they clashed with police, hung Russian flags from the buildings and called for a referendum on independence.
Ukraine’s acting president called an emergency security meeting in response.
The unrest comes amid tensions between Russia and Ukraine over the removal of pro-Moscow President Viktor Yanukovych and Russia’s annexation of Crimea.
Russian President Vladimir Putin has said Moscow has the right to protect the Russian-speaking population there.
Ukraine’s leaders deny the country’s Russian speakers are under threat and have said they will resist any intervention in their country.
Ukrainian Acting President Olexander Turchynov cancelled a planned visit to Lithuania and called a meeting of the country’s security chiefs to deal with the unrest.
In Donetsk, in what was reportedly the day’s most violent protest, a large group of activists broke away from a crowd rallying in the main city square to attack and occupy the regional government seat.