French President Nicolas Sarkozy has said the European Union will not be able to expand further without ratification of the Lisbon treaty, reported BBC.
An EU summit in Brussels has been overshadowed by divisions over the reform treaty - which was rejected by Irish voters in a referendum last week.
A decision on what to do next is expected to be postponed until October.
The EU leaders, who continue talks on Friday, did agree to scrap diplomatic sanctions against Cuba imposed in 2003.
Mr Sarkozy's comments about EU expansion came after clear divisions emerged over the future of the treaty and the meeting decided that the Irish should report back in October on possible ways out of the impasse.
"Without the Treaty of Lisbon there won't be any enlargement," he said.
"You can't say no to reforms and yes to enlargement."
The BBC's Jonny Dymond says one diplomat described Mr Sarkozy's comments as a threat - and the wrong signal to send to the Irish people - and those of Croatia and Turkey, who hope to join the 27-member bloc.
Mr Sarkozy did add that he would travel to Ireland when France takes over the rotating EU presidency in July to help find a solution.
The treaty must be ratified by all member states to take effect. Nineteen have approved it so far, the latest of which was the UK, where it passed through parliament on Wednesday.
European Commission President Jose Manuel Barroso urged respect for the Irish "No" vote but said he was confident all the countries would complete the ratification process. He ruled out renegotiating the reform treaty.
"When a treaty is signed by 27 governments it's not just for fun," he said. "It's inconceivable that a government signs a treaty without the intention of ratifying it. It's a principle of international law."
Slovenian Prime Minister Janez Jansa, whose country is the current holder of the EU presidency, said enlargement should not be a victim of the delay in ratifying the treaty.
"I believe we'll find a way out of the situation before any candidate country is ready for a decision on accession to be taken in the EU," he said. "I don't think the commission wants to slow down the process."
Germany rejected the idea of moving ahead on Lisbon without Ireland, which won strong Czech backing at the summit.
Our correspondent says concern is swirling around the intentions of the Czech government.
Pressed on whether he intended to ratify the treaty, Czech Prime Minister Mirek Topolanek said that he was not going to put the brakes on to halt ratification.
But he added he would not bet 100 crowns (£3; $6) on a "Yes" from parliament.
Mr Barroso has also been pressing for the two-day summit to focus on other issues such as fuel and food prices. After the first day of the summit, he announced:
* an emergency package for fisheries, amounting to 30,000 euros (£23,600) per vessel
* An extended programme to distribute food aid inside the EU to the most deprived people, increasing the aid from 300m euros (£236m) a year to 500m euros (£39m) a year
* A new EU fund to help the agricultural sector in developing countries
At the summit, the leaders will also turn their attention to Zimbabwe's presidential run-off vote.