Downing Street confirmed on Friday night that Mr Johnson had received a legal questionnaire from Metropolitan Police officers investigating events in No 10.
The Prime Minister now has seven days to adequately explain his attendance or face a fine for breaking his own Covid regulations, which would deepen this crisis surrounding his leadership.
Scotland Yard says the questionnaires ask for an “account and explanation of the recipient’s participation in an event” and have “formal legal status and must be answered truthfully”.
A No 10 spokeswoman said: “We can confirm the Prime Minister has received a questionnaire from the Metropolitan Police. He will respond as required.”
Mr Johnson has denied any wrongdoing, but he is alleged to have been at up to six of the 12 events being investigated as part of Operation Hillman.
Downing Street would not say whether the letter was questioning the Prime Minister over one or more events.
Among them, Mr Johnson has admitted attending a “bring your own booze” party in the No 10 garden in May 2020 during the first lockdown, but insisted he believed it would be a work event.
He also allegedly attended a gathering organised by his wife Carrie Johnson in the official Downing Street residence, during which Abba songs were reportedly heard on the November 2020 night of Dominic Cummings’ departure as chief adviser.
Mr Cummings tweeted: “great work tory mps leaving this crippled joke of a pm spending next 7 days bunkered down with lawyers trying to remember all his different lies while another major global crisis unfolds.”
More than 50 individuals across Downing Street and wider Government are expected to receive questionnaires.
Outgoing Met Police Commissioner Dame Cressida Dick this week suggested some of those being contacted by officers will end up with fines.
“Clearly, some, but probably not all, of those people may very well end up with a ticket,” she told BBC Radio London.
The development further imperils the embattled Prime Minister.
Former Conservative leader Sir Iain Duncan Smith has said it would be “very tough” for Mr Johnson to cling on to power if he is fined.
“It will be difficult, he knows that,” the senior MP said in an interview with the i newspaper.
Sir Iain added: “If you’ve set the laws, and you break them and the police decide you have broken them… and then there’s the unredacted (Sue Gray) report – the two things will come together.”
Fifteen Tory MPs have publicly called for Mr Johnson to go, while more are thought to have privately written to the 1922 Committee of backbench Tories calling for a no confidence vote.
More still are poised to do so if the Prime Minister is found to have broken his own coronavirus laws, or further damaging details emerge from the Sue Gray inquiry.
He will face a vote of no confidence if 54 Conservatives write to 1922 Committee chair Sir Graham Brady, and would be ousted if more than half of his MPs subsequently voted against Mr Johnson.
Tobias Ellwood, one of the Conservative MPs to have written a letter, told BBC Newsnight: “We need to get this resolved. The nation is looking at this. There’s a massive level of trust to be regained. We need to move forward, this is a mess.”
The questionnaire was also dispatched during a difficult time for the Met, with Dame Cressida having been pressured into announcing her resignation after a series of scandals.
Her replacement will be chosen by Home Secretary Priti Patel, though London Mayor Sadiq Khan’s views should also be consulted.