The Eurosceptic Czech president opposes the treaty
The upper house of the Czech parliament has voted to approve the European Union's Lisbon Treaty, removing one of its few remaining obstacles.
Czech ratification will not be complete until signed by President Vaclav Klaus, a Eurosceptic who appears in no rush.
But the spotlight is now on the Republic of Ireland, where a second referendum is seen as the last major hurdle in the treaty's path.
The treaty cannot take effect unless all 27 EU member states ratify it.
The Czech Senate voted 54-20 in favour of the reform treaty, aimed at streamlining EU institutions to make them more flexible after the 27-nation bloc's enlargement in recent years.
Its passage depended on many members of the conservative Civic Democratic Party (ODS) led by Czech Prime Minister Mirek Topolanek, putting their doubts aside and backing the plan.
LISBON TREATY PROGRESS
Approved by parliament: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Lithuania, Luxembourg, Malta, The Netherlands, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, UK
Defeated by referendum: Irish Republic
Delays: Legal challenge holding up ratification in Germany, Polish and Czech presidents also resisting ratification
Some Civic Democrat senators have already vowed to send the treaty back to the constitutional court.
The treaty is seen by opponents as a way to impose a federalist agenda, undermining national sovereignty.
Czech President Klaus - perhaps the treaty's highest profile opponent - has not said when he will sign it.
"The Czech government and parliament cannot wield any influence whatsoever on the president. He is free to decide as he wishes," said Senate chairman Premysl Sobotka.
The government seemed relieved that the long ratification process was over - not least since the issue has hung over the Czechs' current presidency of the EU
Alexandr Vondra, Czech deputy prime minister for European affairs, said: "This is an important day for the Czech Republic, for its position and influence in the EU and the world."
European Commission chief Jose Manuel Barroso said: "This is very good news. I am very happy at the approval today."
Only the Czech Republic, Poland, Germany and Ireland have yet to complete ratification of the treaty.
Poland's President Lech Kaczynski, another Eurosceptic, says he will not sign the treaty until it is passed in Ireland, even though the Polish parliament has approved it.
In Germany legal objections are delaying ratification, though parliament has passed it.
In Ireland, where voters rejected the treaty in a referendum last June, the government plans to hold a second vote this year after securing sovereignty "guarantees".
Ireland was the only EU member state to hold a referendum on Lisbon. Other governments argue that it is an amending treaty which does not change the EU enough to justify a referendum.