Unvaccinated players will not be allowed to compete at the 2022 Australian Open, says tournament director Craig Tiley.
There had been confusion over the issue in recent months with contradictory statements from leading Australian politicians.
Defending men's champion Novak Djokovic has said he does not want to publicly reveal his vaccination status.
"Novak knows he will have to be vaccinated to play," Tiley said.
"We would love to have him here."
Tiley also confirmed that the tournament, which runs from 17-30 January in Melbourne, would be played in front of capacity crowds.
A significant number of tennis players remain unvaccinated. In late October, 35% of those on the men's ATP Tour were unvaccinated. The Women's Tennis Association (WTA) is yet to provide BBC Sport with the vaccination uptake percentage among its players.
The WTA players' council sent a letter to players in October stating it had been told all competitors would be able to travel to Melbourne.
The letter also stated that all players, regardless of vaccination status, would have to show proof of a negative test within 72 hours of departure.
However, Victoria Premier Daniel Andrews said in October that he did not think an unvaccinated tennis player would be given a visa to enter Australia.
Australia has had some of the strictest restrictions on travel and movement during the pandemic.
Players, officials and support staff who travelled for the 2021 tournament were required to complete a 14-day quarantine period on their arrival in Australia.
Some were not allowed to leave their rooms after coronavirus cases were detected on their flights, leading them to set up makeshift practices in their rooms.
England's men's cricket team, who are currently in Australia for the Ashes, spent two weeks in quarantine at a Gold Coast resort before they could begin fully their preparations.
Photo Getty Images Caption: Defending men's champion Novak Djokovic has said he does not want to reveal his vaccination status