Poland, the biggest of 10 mainly ex-communist countries on course to join the EU on May 1, 2004, follows Malta, Slovenia, Hungary, Lithuania and Slovakia, which staged positive referenda on entry.
Although a Yes to membership is widely expected, 14 years after Poland led eastern Europe's breakaway from communism, all eyes will be on turnout.
More than half of registered voters must cast their ballots to validate the referendum.
Polling stations were due to open tomorrow and Sunday 04:00 GMT, closing 14 hours later at 18:00 GMT.
First halfway turnout figures were expected tomorrow from 20:00-22:00 GMT, with first results expected from 20:00 GMT on Sunday.
The latest poll released yesterday encouraged Warsaw's EU membership hopes, showing that 80,9% of voters would say Yes, while 68% would definitely take part in the referendum.
According to the poll by the PBS institute on 1 074 people on June 4, of those who have decided to vote 13% plan to say No and 6,1% have not yet decided.
But with Poland's voters traditionally lazy about going to the polling stations, turnout has been a real headache for the Warsaw government.
Less than half turned out at local elections late last year, and at the only two referenda held since the end of communism in 1989.
The government's turnout worries were increased by alarmingly low turnouts of 45,6% and 52% in the referenda in Hungary and Slovakia.
If the worst were to happen and the referendum turned out to be invalid, the buck would pass to the two houses of parliament.
Dominated by pro-European parties they would confirm Poland's EU membership, but Poland would enter the EU on May 1, 2004 with what government spokesperson Michal Tober called "a bad taste in your mouth".
As referendum day loomed, analysts believed voters would rise to the challenge.
"The question is, how many players will play this game," Marek Sarjusz-Wolski, a journalist at Unia and Polska magazine and member of Poland's European integration council, said.
"I'm sure it will be over 50%," he said.
"I think that there will more than 50%, but I don't think that it will be much more than 54-55%," Lena Kolarska-Bobinska, the director of the Institute of Public Affairs in Warsaw, said.
The referendum comes at a bad time for the Warsaw government, deeply unpopular after being rocked by a corruption scandal that has indirectly implicated Prime Minister Leszek Miller.
Struggling along in a minority government since early March Miller has also been pilloried for his economic policy, in spite of a recent turn around, and for having the highest unemployment rate in the OECD: more than 18% in April.
As referendum day loomed, pleas have poured in from around Europe and across the Atlantic, as foreign leaders offered moral support.
"I hope very much for the sake not just of Poland also for the sake of the whole of Europe that Poland votes Yes," British Prime Minister Tony Blair said, as he passed through Warsaw to thank Poland for fighting in the Iraq war.
But the most eagerly awaited words, and what observers say are the most influential for the overwhelmingly Roman Catholic country, came from the Vatican as Poland's beloved John Paul II weighed in.
"Europe needs Poland. The Church in Europe needs the Polish people's witness to the faith. Poland needs Europe," he added, addressing an estimated 20 000 Polish pilgrims in a special audience in St Peter's Square. – Sapa-AFP.
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