As a rapid-acting dissociative anesthetic, ketamine has been used in drug-facilitated crimes. The aim of this study is to investigate the disposition of ketamine and its main metabolite norketamine in hair after a single dose of ketamine. Four healthy volunteers were recruited into the study. Hair was collected 1, 2, 3, 4, 8, 12 and 16 weeks after a single oral dose of ketamine solution (10 mg) and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry. The wet cotton swab wiped the scalp of the subjects at 1 h, 24 h, 48 h and 1 week after administration. Maximum hair concentrations (C (max)) for ketamine and norketamine were 19.0 ± 6.5 and 18.7 ± 13.3 pg/mg, respectively. Except for the first week, the ratio of ketamine to norketamine in most of segments (87.5%) was greater than 1. All the cotton swab samples collected at 24 and 48 h were positive. The results from cotton swabs and the concentrations of ketamine and norketamine in hair segments collected at different times showed that some of ketamine and norketamine incorporated into hair originated from sweat and sebum on the scalp of the subjects.