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A photo of Boris Johnson, his wife and 17 staff members in the Downing Street garden with bottles of wine and a cheese board shows them having a "work meeting", No 10 has said.
The Guardian, which published the image, says the gathering took place in May 2020, during the first lockdown.
At the time, people could only meet one person from another household outside.
In response, No 10 said: "Work meetings often take place in the Downing Street garden in the summer months."
"On this occasion there were staff meetings following a No 10 press conference," a spokesperson added.
"Downing Street is the prime minister's home as well as his workplace. The prime minister's wife lives in No 10 and therefore also legitimately uses the garden."
Labour's deputy leader Angela Rayner urged Mr Johnson to "tell us the truth" about gatherings at Downing Street.
SNP deputy Westminster leader Kirsten Oswald called for a "judge-led inquiry" to determine what happened.
The photograph shows a gathering of staff in the Downing Street garden following a news conference held by then-Health Secretary Matt Hancock on 15 May 2020, according to the Guardian.
The newspaper pointed out that Mr Hancock used that news conference to warn the public to stick to social-distancing rules despite the warm weather.
He said at the time: "People can now spend time outdoors and exercise as often as you like and you can meet one other person from outside your household in an outdoor, public place. But please keep two metres apart.
"This weekend, with the good weather and the new rules, I hope people can enjoy being outside but please stick with the rules, keep an eye on your family and don't take risks."
Responding to previous reports of a 15 May gathering, a spokesperson for Mr Hancock said he left the building at 18:30 GMT after speaking to Mr Johnson in the garden.
"There is no suggestion that Mr Hancock did anything wrong," his spokesperson added.
Senior civil servant Sue Gray has been tasked with completing a review into a number of alleged gatherings inside government buildings in November and December 2020 - against rules at the time.
Ms Gray replaced cabinet secretary Simon Case, who stepped down from the review after it was revealed he had known about a quiz held in his office.
The government Christmas parties of 2020
13 November 2020: Sources have told the BBC there were impromptu drinks to mark the exit of the former director of communications at No 10, Lee Cain - but it was staff having drinks at their desks and was over by 20:30 GMT.
13 November 2020: Also the date Dominic Cummings left as chief adviser to the PM. Sources told the BBC that several Downing Street staff members attended a gathering with Carrie Johnson in the flat where the prime minister and his wife live above No 11 after exits. A source said music was blaring and could be heard elsewhere in the building. But others said to be at the party denied it took place, as did a spokeswoman for Mrs Johnson.
25 November 2020: The Times reported Treasury officials held a drinks party in their office to celebrate Chancellor Rishi Sunak's spending review during the lockdown. The newspaper said two dozen civil servants were understood to have attended the event. But a spokesman for the department insisted it was an "impromptu" event, with a "small number" of staff who celebrated around their desks.
27 November 2020: Another No 10 aide, Cleo Watson, left Downing Street marked by an event. Again, sources said it was not formally organised, but people were having drinks and Mr Johnson made a speech.
10 December 2020: The Department for Education has confirmed it had a gathering in the office to thank staff for their work during the pandemic. Drinks and snacks were brought by those who attended and no outside guests or support staff were invited.
14 December 2020: The Conservatives have confirmed a report in the Times that there was an "unauthorised social gathering" in the basement of their party's building, held by the team of the London mayoral candidate at the time, Shaun Bailey. It was described as "raucous", with No 10 aides
15 December 2020: Multiple sources told the BBC there was a Christmas quiz for No 10 staff in the Cabinet Office, with invites sent out in advance via email, telling people to form teams of six. Downing Street have said it was a "virtual gathering" but while some joined in that way, sources said there were groups in the room sat in their teams. This is the event the Sunday Mirror released a picture of the PM attending virtually.
18 December 2020: A Downing Street Christmas party took place on this date, as first reported by the Mirror. A source told the BBC there was food, drinks and games at the gathering that went on past midnight.
DWP gatherings: The Department for Work and Pensions confirmed a "core team" of staff drank alcohol and ate takeaways "on a number of occasions" whilst working late in the office while Covid restrictions were in place. It followed a report in the Sunday Mirror alleging political staff and officials working for minister Therese Coffey frequently drank after work until the early hours of the morning.