Polish President Lech Kaczynski has signed the European Union's much-delayed Lisbon Treaty.
His signature means the treaty, which is intended to streamline decision-making, remains unratified by only one country, the Czech Republic.
It must be ratified by all 27 member states before it can come into force.
Mr Kaczynski, a noted Eurosceptic, said he was convinced the treaty would be successful but that the EU should remain a union of sovereign states.
The treaty's prospects of coming into force received a major lift last week when Irish voters approved it in a second referendum.
But the Czech President Vaclav Klaus then raised fresh doubts when he said he would not sign the treaty unless his country was granted an opt-out from the EU's Charter of Fundamental Rights.
Mr Klaus raised fears about possible property claims by Germans expelled from the then Czechoslovakia after World War II.
Mr Kaczynski said in a speech just before the signing he was "deeply convinced" that the "great experiment" of the treaty would be successful.
Gavin Hewitt, BBC News, Brussels
There is an element of farce creeping into the drama of the Czech Republic and the Lisbon Treaty. We now know what President Klaus's conditions are for signing the treaty - or rather I think we do.
To start with, he did not intend to reveal his hand. He feels he was pushed into it. The Czech president is clearly irritated that, in his view, the Swedish prime minister disclosed a confidential conversation they had.
"The fact that the Irish people changed their minds meant the revival of the treaty, and there are no longer any obstacles to its ratification," he said.
"Today is a very important day in the history of Poland and the European Union."
But he said that the EU was a "union of sovereign states" and should remain so, adding that it remain open to new members such as countries of the former Yugoslavia and Georgia.
European Commission President Jose Manuel Barroso, European Parliament speaker Jerzy Buzek and Swedish Prime Minister Fredrik Reinfeldt, whose country holds the EU's rotating presidency, attended the Polish ceremony.
Welcoming the Polish signing, Mr Reinfeldt said that the EU "eagerly awaits" the full ratification of the treaty.
"We do not need more delays," he said.
The BBC's Jonny Dymond in Brussels says that Mr Kaczynski's signature leaves the Czech Republic, and in particular its Eurosceptic president, entirely isolated.
But, he says, that appears not to concern Mr Klaus.
The president is insisting on a "footnote" being added to the text so as to exempt his country from the Charter of Fundamental Rights much like Poland and the UK already are, our correspondent says.
"Before ratification, the Czech Republic must, additionally at least, negotiate a similar exemption," Mr Klaus was quoted by the Reuters news agency as saying on Friday. "I believe that this exemption can be resolved quickly."
He said the charter would "allow [claimants] to circumvent Czech courts and place, for example, property claims by people expelled after World War II directly at the European Court of Justice".
President Klaus has compared EU institutions to the old Soviet bloc
Mr Klaus has said previously that he will not sign the treaty until the Czech Constitutional Court rules on a new legal complaint against it, lodged by senators allied to him.
The Czech parliament has already approved the treaty and his critics note that it is the government, not the president, who negotiates international treaties.
Nevertheless, President Klaus's move has already had an impact in Brussels, our correspondent adds.
Officials say the next summit at the end of October will now probably be devoted solely to dealing with Czech objections, rather than selecting people for the new posts created by the Lisbon Treaty.
France has already expressed opposition to Mr Klaus's demand.
Supporters of the treaty are also concerned that if Mr Klaus holds up the treaty, Britain's opposition Conservatives may fulfil their pledge for a referendum on it should they come to power.
A UK general election must be held by June next year.
At the Conservative party conference this week, shadow foreign secretary William Hague restated its opposition to the treaty, saying the EU should not place its own president above any nation's.
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