The Metropolitan Police has started dedicated traffic patrols on the M4 Olympics Games Lane in west London to enforce restrictions.
The 3.5-mile lane between Heston and Brentford is reserved for official Olympics vehicles. Other drivers using it will face a £130 fine.
The restrictions began on Monday, but until Tuesday night it was unclear who would monitor and fine transgressors on the route maintained by the Highways Agency.
Apart from official Olympics vehicles, only black cabs are allowed to use the lane, which forms a part of the 30 miles of Olympic Route Network (ORN) in London.
The restrictions on the lane came into force at 05:00 BST on Monday as athletes began arriving at Heathrow Airport.
A Highways Agency spokesman told the BBC: "We are asking motorists not to use the Games Lane while it is in operation.
"To ensure that it continues to operate well the Metropolitan Police will be enforcing compliance on the M4 Games Lane from today (Tuesday)."
A Met spokesman said: "The Highways Agency and Transport for London have today (Tuesday) asked the Metropolitan Police Service to enforce compliance on the M4 Games Lane.
"There are now dedicated traffic patrols."
The restrictions on the rest of the Games Lane on the ORN, which falls on roads managed by TfL, will begin from 25 July.