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With 92% of the referendum votes counted the "no" campaign had a clear margin of 52.4% to 47.6% for the "yes" vote. Turnout was estimated at 80%.
The result is a blow for Prime Minister Gro Harlem Brundtland, who has made Norwegian membership of the EU her main political goal for the last four years.
"It was the people who made the decision, and we as a country have to live with that," she said.
Norway first rejected membership of the European Economic Community, as the EU was then known, in 1972. That result, by a similar margin to today's vote, forced the resignation of the government.
Mrs Brundtland has ruled out resignation this time, but it remains unclear how she will lead Norway into a non-European future.
The leader of Norway's Anti-European Union campaign, Anna-Enger Lahnstein, told supporters "With this, we have said Yes to Europe and Yes to international solidarity - but we don't want to join the Union."
The result threatens to leave the country bitterly divided after the closely-fought campaign.
Voters in the far north, the stronghold of the "no" campaign, rejected membership by a margin of 80-20, whereas the capital, Oslo, where a third of voters live, backed membership.
The "no" campaign led consistently in opinion polls throughout the campaign, but last-minute gains by the "yes" campaigners narrowed the gap and made the result too close to predict.
Recent votes for EU membership in fellow Scandinavian countries Finland and Sweden swayed many voters, but even that was not enough to reverse the trend.
The "yes" campaign has centred on the potential for Norway to become isolated if it doesn't join neighbouring EU countries in an era of expansion and change.
However, the "no" campaign argued EU membership would undermine the country's independence and its control over its rich petroleum and other natural resources.
But economists also warn that although Norway's oil-dependent economy is strong in the short term, North Sea oil and gas revenues are due to tail off soon and growth is expected to weaken.
The final result was 52.2% no to 47.8% yes, with turnout a record 88.6%.
A poll in 2002, following the introduction of the Euro, suggested the tide had turned in Norway, and more than 50% were now in favour of EU membership.
However, there are no imminent plans to reconsider applying.
Gro Harlem Brundtland resigned two years after the "no" vote, after three terms in office.
She served as director of the World Health Organisation from 1998-2003.
In 2004 she was listed by the British newspaper, the Financial Times, as the 4th most influential European for the last 25 years, behind the Pope, Mikhail Gorbachev and Margaret Thatcher.
In May 2004 the EU took in 10 new members, expanding it to 25 member nations, with more under consideration.
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