Ukraine's president has hailed a "historic" trade pact with the European Union, calling it the "most important day" since independence in 1991.
But Russia said the partnership agreement, also signed by two other ex-Soviet states Georgia and Moldova, would have "serious consequences".
The pact is the issue that triggered Ukraine's current crisis.
Russian President Vladimir Putin said making Ukraine choose between Russia and the EU would split it in two.
A week-long ceasefire between Ukrainian forces and pro-Russian separatists in restive eastern Ukraine expired on Friday at 22:00 (19:00 GMT).
Rebel spokesmen had indicated they were willing to extend it to 30 June.
Ukrainian President Petro Poroshenko, in Brussels to sign the pact, had said he would take a decision on an extension on Friday evening, after he arrived back in Kiev.
Analysis: Steve Rosenberg, BBC News Moscow
There is a general sense of irritation or perhaps even anger here that Moscow has failed to convince countries like Ukraine, Moldova and Georgia not to sign this historic free trade deal today with the EU.
Moscow has economic concerns about these deals - it is worried that the Russian market could be flooded by cheap goods from the EU that would hit Russian producers.
More pressing for Moscow are the geopolitical concerns here - the whole idea of former Soviet states, countries that Moscow still views as being within its sphere of influence, drifting towards Europe and one day possibly becoming part of the EU - that really grates with Moscow, particularly in the case of Ukraine.
There's a lot of concern about what could happen in eastern Ukraine - the ceasefire announced a few days ago by Mr Poroshenko, and the ceasefire announced by armed separatist rebels, is due to expire today. It's unclear how things are going to develop later.
Russian Foreign Minister Sergei Lavrov had said he would welcome an extension, but not if it were simply an ultimatum for separatists to lay down their arms.
Mr Putin insisted on a long-term ceasefire to allow for negotiations between the Ukrainian government and separatists, urging Mr Poroshenko to embark on a "path of peace, dialogue and accord".
Mr Putin said: "There is bloodshed in the south-east Ukraine, humanitarian catastrophe, tens of thousands of refugees have to look for shelter, on Russian territory."
Mr Putin said that "attempts to force on the Ukrainian people an artificial choice between Europe and Russia brought [a split] to society, a painful internal confrontation".
The refusal of Mr Poroshenko's predecessor, Viktor Yanukovych, to sign the EU deal, under pressure from Russia, had led to protests in Kiev and his eventual overthrow this year.
In Brussels, Mr Poroshenko hailed the 1,200-page Association Agreement as a turning point, describing it as a "symbol of faith and unbreakable will".
"What a great day! It is a historic day, maybe the most important day since independence," he said.
Mr Poroshenko also said he saw the signing as the start of preparations for joining the EU bloc.
The pact binds the three countries more closely to the West both economically and politically.
European Council President Herman Van Rompuy described the pacts as a "great day for Europe".
But Russian Deputy Foreign Minister Grigory Karasin said there would "undoubtedly be serious consequences for Ukraine's and Moldova's signing".
The Kremlin immediately said it would take "all the necessary measures" against Ukraine.
Russia has warned it will hit Ukraine with punishing trade restrictions.
It could withdraw Ukraine's duty-free benefits as a member of the Commonwealth of Independent States.
The European Council on Friday also issued a policy statement on Ukraine, setting out key steps it expected to happen by Monday, including the return of three key checkpoints to Ukrainian forces and the "launch of substantial negotiations on the implementation of President Poroshenko's peace plan".
Mr Poroshenko set out a 15-point peace plan on 20 June. It involves decentralising power and holding early local and parliamentary elections.
It also proposes the creation of a 10km (six-mile) buffer zone on the Ukrainian-Russian border, and a safe corridor for pro-Russian separatists to leave the conflict areas.
German Chancellor Angela Merkel warned Russia the EU was prepared for "drastic measures" if there was no speedy progress on Mr Poroshenko's peace plan.
French President Francois Hollande said more measures would be taken if there was no progress after Sunday, when he and Mrs Merkel will speak to Mr Putin by phone.
Fighting is said to have continued in some areas of eastern Ukraine despite the ceasefire.
But rebels have now released four international observers captured more than a month ago.
More than 420 people have been killed in fighting between pro-Russia rebels and government forces in eastern Ukraine since mid-April, the UN estimates.
The separatists have declared independence, claiming that extremists have taken power in Kiev.
Their move followed Russia's annexation of Ukraine's Crimea region.