All lanes of the eastbound 401 were closed in Cobourg, east of Regional Road 45 to Grafton, following a serious multi-vehicle crash on Tuesday.
The collision involved two tractor-trailors and up to eight vehicles.
Two air ambulances were called to take two victims to St. Michael's Hospital in Toronto. While several others were taken to a local hospital.
The highway was closed for several hours.
Motorists were being advised to get off the eastbound Hwy. 401 at Port Hope.