An estimated 46,499,537 people are entitled to take part in the vote.
Polling stations opened at 07:00 BST and will close at 22:00 BST.
It is only the third nationwide referendum in UK history and comes after a four-month battle for votes between the Leave and Remain campaigns.
The referendum ballot paper asks the following question: “Should the United Kingdom remain a member of the European Union or leave the European Union?”
Whichever side gets more than half of all votes cast is considered to have won.
After the referendum polls close, sealed ballot boxes will be collected and transported to the count venue for each of the 382 local counting areas.
These represent all 380 local government areas in England, Scotland and Wales, plus one each for Northern Ireland and Gibraltar.
Individual areas’ results will then be declared throughout the night, along with results from 11 regional counts.
Depending on how close the poll is, the result may become clear before the final national result is officially declared by the Chief Counting Officer, who will be based at Manchester Town Hall.
The Electoral Commission estimates a final result “around breakfast time” on June 24.
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