Venice and the surrounding region are voting on whether to break away from Italy.
Recent opinion polls suggest that two thirds of the 4 million electorate favor splitting from Rome, but the vote will not be legally binding.
The poll was organized by local activists and parties, who want a future state called Republic of Veneto.
This would be reminiscent of the sovereign Venetian republic that existed for more than 1,000 years.
A focal point for culture, architecture and trade, Venice lost its independence to Napoleon in 1797.
Online voting is due to continue until Friday.
The vote received very little coverage in Italy’s national media but the organizers said they expected as many as two million people to take part.
It also reflects a growing separatist mood in parts of Europe, such as Spain’s Catalonia region and Scotland, which votes on whether to become independent in September.
Moves towards independence often evoke more sympathy in wealthy northern Italy, where many resent what they see as the poorer south’s waste and corruption.
Luca Zaia, governor of Veneto, the Venice region, rejected suggestions that the Italian constitution would prevent secession.