Doncaster Racecourse has been told by local health officials to stop spectators attending its St Leger meeting after Wednesday's opening day.
"On the grounds of public health and public safety I have instructed the course to hold the St Leger Festival behind closed doors from tomorrow," said Dr Rupert Suckling, director of Public Health for Doncaster.
More than 2,500 spectators are expected at Doncaster on Wednesday as the Leger meeting starts amid uncertainty over new government rules.
It is the first crowd at a British horse racing fixture in six months as part of a government pilot scheme for sporting events.
Arena Racing Company (Arc), which runs Doncaster Racecourse, confirmed the remainder of the four-day meeting will be held without spectators.
"This pilot event represents a hugely important step not just for our business but for the whole of British racing as well as the sports and hospitality industries as a whole." said Mark Spincer, the managing director of Arena's racing division.
"We do, of course, fully understand and respect the decision and will be contacting all of our customers booked for the remaining three days as a matter of urgency."
Spectators were allowed at Doncaster on the first day of the St Leger meetingThe government said on Tuesday night that social gatherings in England would reduce from a maximum of 30 to six people from Monday in response to rising numbers of coronavirus cases.
Health Secretary Matt Hancock said more details will be announced on Wednesday.
"The Prime Minister is going to set out more details on the consequences of new rules for six people gathering later today. We'll set out what this means for some of these events we were planning to do," he told BBC Breakfast.
Prime Minister Boris Johnson is due to expand on the new rules at a Downing Street news conference at 16:00 BST, where he will be joined by Professor Chris Whitty, England's chief medical officer, and Sir Patrick Vallance, the UK government's chief scientific adviser.
Doncaster Central MP Rosie Winterton lobbied for the meeting to be held with a limited attendance as one of the government's pilot sporting events, but the town's mayor, Ros Jones, has said the risk of holding the fixture with crowds is "too great".
Racing has been held behind closed doors since resuming on 1 June after a 10-week suspension because of the Covid-19 pandemic.
Up to 3,640 people will be permitted entry on Wednesday, and the racecourse says more than 2,500 tickets have been sold. A limit of 6,202 was planned on the other days, including Saturday, when the Leger, the world's oldest Classic race, is staged.
Racegoers had to sign up to a code of conduct and have been split into dedicated zones, with social distancing protocols in place.
More than 54,000 spectators attended the four-day meeting last year, including 27,000 on the Saturday.
A trial attendance of 5,000 people was due to take place on the fifth and final day of Glorious Goodwood last month, but that was scrapped at the last minute after a spike in coronavirus cases across parts of Britain.
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