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King's Highway 401, also known by its official name as the Macdonald–Cartier Freeway and colloquially as the four-oh-one, is a 400-series highway in the Canadian province of Ontario stretching 817.9 kilometres (508.2 mi) from Windsor to the Quebec border. The segment of Highway 401 passing through Toronto is the busiest highway in North America, and one of the widest and busiest in the world. Together with Quebec Autoroute 20, it forms the road transportation backbone of the Quebec City–Windsor Corridor, along which over half of Canada's population resides. The entire route is maintained by the Ministry of Transportation of Ontario (MTO) and patrolled by the Ontario Provincial Police. The posted speed limit is 100 km/h (62 mph) throughout its length.

Three individual highways were renumbered "Highway 401" by the end of 1952: the partially completed Toronto Bypass between Weston Road and Highway 11 (Yonge Street); Highway 2A between West Hill and Newcastle; and the Scenic Highway between Gananoque and Brockville, now known as the Thousand Islands Parkway. These three sections of highway were 11.8 km (7.3 mi), 54.7 km (34.0 mi) and 41.2 km (25.6 mi) long, respectively, at the time of their assumption to provincial highway status. Highway 401 became fully navigable from Windsor to the Quebec border in 1964. The following year, it was given a second designation, the Macdonald–Cartier Freeway, in honour of the fathers of Confederation. By the end of 1968, the Gananoque–Brockville section was bypassed and the final intersection grade-separated near Kingston, making Highway 401 a freeway for its entire 817.9-km length. On August 24, 2007, the portion of the highway between Glen Miller Road in Trenton and the Don Valley Parkway / Highway 404 Junction in Toronto was designated the Highway of Heroes, as the road is travelled by funeral convoys for fallen Canadian Forces personnel from CFB Trenton to the coroner's office in Toronto.

In 2011 construction began on a westward extension of Highway 401 that will be known as the Right Honourable Herb Gray Parkway. This new route will generally follow, but not replace, former Highway 3 between the current end of the freeway and the E. C. Row Expressway, at which point it will turn and follow that route to the Detroit River International Crossing.

Elsewhere in Ontario, plans are underway to widen the remaining four lane sections between Windsor and London to six lanes and to widen the route between Cambridge and Milton as well as through Oshawa. The expansive twelve-plus lane collector–express system will also be extended west through Mississauga to Milton and east through Ajax and Whitby.