It travels in a southeast-to-northwest path from the Virginia state line near Pennington Gap to the Indiana state line, on the Ohio River at Milton, Kentucky and Madison, Indiana.

At Hyden, US 421 turns westward towards Clay County, and crosses the Hal Rogers Parkway via an overpass without access near [[Manchester, ( 421 actually goes through Manchester Ky to Hal Rodgers pkwy.)

It traverses the far northeastern corner of Rockcastle County before entering Madison County where it joins U.S. 25 south of Richmond near the Blue Grass Army Depot.

After Exit 99, the two U.S. routes continue to run concurrently until downtown Lexington just after crossing US 27/60/68.

While US 25 makes a right turn onto Georgetown Road, US 421 continues straight northwestward, as Leestown Pike, towards Midway and Frankfort.