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It's the second consecutive defeat for the proposed charter. Three days earlier, French voters rejected the constitution that would have created a political and economic entity rivalling the United States.
Calling the results disappointing, Dutch Prime Minister Jan Pieter Balkenede conceded defeat, saying he would respect the will of the Dutch people. The country's parliament will meet Thursday to discuss the results.
The outcome was expected. Most opinion surveys leading up to Wednesday's referendum suggested it would be rejected despite the campaigns of the mainstream Dutch parties, including the government, who advocated a Yes vote in the referendum.
Socialist party supporters celebrate in Amsterdam, Netherlands, after preliminary results of the referendum were announced. (AP photo)
Voter turnout in the nation of 16 million was 62.8 per cent.
- INDEPTH: European Union
While the Dutch result is not legally binding, legislators say they will respect the outcome.
Supporters of the constitution argued it would streamline decision-making in the EU and create a single foreign minister to give Europe more sway in international affairs.
Opponents fear that the Netherlands will be engulfed by a superstate dominated by Germany, France and Britain. They also fear the constitution could end liberal Dutch policies such as tolerating marijuana use, prostitution and euthanasia.
Nine countries have ratified the constitution: Austria, Hungary, Italy, Germany, Greece, Lithuania, Slovakia and Slovenia by votes of their parliaments; and Spain by a referendum.
The proposed constitution needs support from all 25 nations.
With Sunday's referendum result, France became the first EU member to reject the constitution. About 55 per cent voted against it in a legally binding referendum that saw a turnout of 70 per cent of the country's registered voters.
German Chancellor Gerhard Schroeder, a strong supporter of the charter, said the French and Dutch votes must be acknowledged.
"The crisis surrounding the ratification of the European constitution must not become Europe's general crisis," said Schroeder.
EU Commission President Jose Manuel Barros urged European leaders to avoid making hasty decisions until an EU summit in mid-June.
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