Boris Johnson has been given until Monday to explain why a letter from him in regards to his relationship with Jennifer Arcuri should be private, the London Assembly said.
The PM has faced accusations of not declaring a conflict of interest after the US businesswoman was given grants and access to trade trips during his time as London Mayor.
He responded to the London Assembly probe earlier this week, though his letter was marked "private and confidential" and "not for publication".
Mr Johnson, who has denied any wrongdoing in the matter, has previously accused his "old friends" in the Assembly of "barking up the wrong tree" with their investigation.
In the letter to Mr Johnson's solicitors, Len Duvall AM, chairman of the Assembly's oversight committee, said: "Transparency and accountability are at the heart of the London Assembly's operations and I was disappointed to see that your response is marked 'Private and Confidential' and 'Not for Publication'.
"The reason for confidentiality is not given and, on the face of it, is unclear to me.
"I would request that you provide reasons for that confidentiality by 5pm on Monday 14 October 2019, so that the GLA oversight committee can consider appropriate handling of the letter."
Labour has also called for the PM's response to be made public.
The oversight committee has asked Mr Johnson for details and a timeline of all contact with Ms Arcuri.
It asked this include "social, personal and professional" during his time as mayor of London.
The Standard previously reported those involved in the London Assembly investigation were considering whether to make the letter from the PM public, while there was reported anger he was trying to "stonewall" the investigation.
The London Assembly also told the PM it found his response to be "insufficient".
Ms Arcuri used a TV interview this week to deny reports that she received favouritism during his eight-year stint as mayor, though she said they had been a "really good friend".
The Independent Office for Police Conduct is looking into whether it should investigate if Mr Johnson committed misconduct in public office.
Additional reporting by PA.
