Ministry of Transportation officials closed the lane as a precautionary measure after the three-metre-wide crater was reported around 8 a.m. at Hwy. 400 and north of Hwy. 88. The area is about midway between Toronto and Barrie.
Crews are racing to repair the area before long weekend traffic begins and Ontarians head to cottage country.
According to the ministry, the sinkhole spanned from the southbound left lane, across the shoulders into the the northbound left lane.
Both the southbound and northbound lanes on 400 were closed as a result.
The ministry said it believes the sinkhole was caused by a crack in the "box culvert" — a square concrete structure that allows water to flow beneath the highway.
Repairs were expected to be completed by the late evening, and the ministry is aiming to have both lanes reopened by midnight.
There were no injuries or damaged cars as a result of the sinkhole.