US 27 crosses into Kentucky entering the Daniel Boone National Forest in the Strunk community, just south of Pine Knot. Heading north, US 27 turns into a four-lane highway in Burnside. US 27 is the main highway running through Somerset. The route continues as a four-lane highway almost to the Pulaski–Lincoln county line and again has four lanes through Stanford. It veers northeast to Lancaster then northwest to the site of Camp Dick Robinson at Kentucky Route 34, becoming four lanes. Entering Jessamine County, the road crosses the Kentucky River. It westerly bypasses the central business district of Nicholasville. Once in Fayette County, US 27 turns into a six-lane highway passing many shopping destinations in Lexington. Passing The Summit at Fritz Farm, Fayette Mall, and Lexington Green, Nicholasville Road is one of the busiest roads in Lexington. The road becomes South Limestone Street upon reaching Cooper Drive. At UK's Albert B. Chandler Hospital, it turns west on four-lane Virginia Avenue, then joins US 68 (Broadway) for its northeasterly journey through Lexington's central business district and the horse-farm country of Fayette and Bourbon counties. The conjoined routes separate on the north side of Paris, where US 27 splits from the town bypass and heads north to Cynthiana, Falmouth, and Newport. It then crosses the Ohio River into Cincinnati.

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