In order to study the incorporation of sildenafil (SDF) and its N-demethylated metabolite (norSDF) into hair, animal model experiments were carried out. After shaving the back hair, SDF was dosed to two sets of three male dark-agouti pigmented rats (5 weeks old) per each group at 25 mg/kg once a day for 5 successive days with intraperitoneal (i.p.) (set1) and oral administration (set2). The regrown back hair was collected 14 d after the first administration. Three typical extraction methods, using methanol-5 M hydrochloric acid, methanol-trifluoroacetic acid and 1 M sodium hydroxide, were evaluated using the rat hair samples containing SDF and norSDF. Methanol-5 M hydrochloric acid was the best extraction method in terms of high efficiency and reproducibility. The extract was purified using Bond Elut Certify columns and was derivatized withtrimethylsilylimidazole:N,O-bis(trimethylsilyltrifluoroacetamide):trimethylchlorosilane (3:3:2) at 90°C for 30 min. The trimethylsilylated products were analyzed by GC-MS using selected ion monitoring. SDF and norSDF were simultaneously detected in the rat hair. The hair concentrations were 4.9—6.3 (av. 5.8) ng/mg and 15.6—20.3 (av. 17.6) ng/mg for SDF and norSDF, respectively, with i.p. administration, and 2.6—4.1 (av. 3.6) ng/mg and 8.1—10.4 (av. 9.1) ng/mg with oral administration. The hair concentrations of norSDF were about three times higher than those of SDF, and the ratios of both compounds showed no significant difference between i.p. and oral administrations. This method was applied to the scalp hair of two patients who orally took SDF at regular intervals for the treatment of penile erectile dysfunction. The hair concentrations of SDF and norSDF in the two patients were 19.8 and 55.9 ng/mg, and 1.7 and 5.6 ng/mg, respectively.