Venice has become the world's first city to introduce a payment system for tourists.
Signs were set up outside the train station and near an entry footbridge warning visitors they had to pay the new €5 charge before diving into Venice's narrow alleyways.
However, the fee has received a backlash from some residents who said they didn't want to live in a theme park.
Reservations are meant to be made online but there is also a booth on hand for those who don't have smartphones. Although there are no turnstiles at the city gateways, inspectors will be making random checks and issuing fines of between 50 and 300 euros to anyone who has failed to register.
Registration booths for people entering the city of Venice
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Venice has become the first city to introduce the tax
Reuters
The city councillor responsible for tourism and social cohesion Simone Venturini said the scheme would help Venice find "a new balance" between residents and day-trippers, but hundreds of local protesters saw things differently.
Giovanni Andrea Martini, a member of an opposition group in the Town Hall who joined the residents' protests, called it "a sad day because Venice is becoming a museum, a theme park."
Around 20 million people visited Venice last year, a city official said, with roughly half of them staying overnight in hotels or holiday lets - an influx which dwarfs the resident population currently put at around 49,000.
People with hotel reservations and visitors aged under 14 do not need to pay the entry fee, but still need to register beforehand. Residents, students and workers are exempt.
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Protests have been held against the tax
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Resident Cristina Romieri said: "We are against this measure because it will do nothing to stop over-tourism.
"Moreover, it is such a complex regulation with so many exceptions that it will also be difficult to enforce it."
Venice narrowly escaped being placed on UNESCO's "World Heritage in Danger" list last year partly because the UN body decided the city was addressing concerns that its delicate ecosystem risked being overwhelmed by mass tourism.
Besides introducing the entry charge, the city has also banned large cruise ships from sailing into the Venetian lagoon and has announced new limits on the size of tourist groups.
There were concerns the city was being overrun
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Italian tourists arriving earlier this week said imposing another charge on visitors was unfair.
Gabriella Pappada, who came from Lecce in southern Italy, said: "I consider Venice to be the most beautiful city in the world and so to deprive a person on a low budget of the opportunity to come here for an hour or two to enjoy this city is surely a shame for these tourists."