Background: Although hair testing is well established for the assessment of past drug exposure, uncer-tainties persist about mechanisms of drug incorporation into hair and interpretation of results. The aim of this study was to administer methamphetamine (MAMP) under controlled conditions as a model drug to investigate drug incorporation into human hair. Material and methods: Seven volunteers with a history of stimulant use received 4 × 10 mg (low) doses of sustained release S-(+)-MAMP HCl within 1 week, with weekly head hair samples collected by shaving. 3 weeks later, 4 of them received 4 × 20 mg (high) doses. After extensive isopropanol/phosphate buffer washing of the hair, MAMP and its metabolite amphetamine (AMP) concentrations were determined in all weekly hair samples by LC–MS–MS in selected reaction monitoring mode with the undeca- and deca-deuterated drugs, respectively, as internal standards (LLOQ, 0.005 ng mg−1). Results: MAMP Tmaxoccurred from 1 to 2 weeks after both doses, with Cmaxranging from 0.6 to 3.5 ng mg−1 after the low and 1.2 to 5.3 ng mg−1 after the high MAMP doses. AMP Cmaxin hair was 0.1–0.3 ng mg−1 and 0.2–0.5 ng mg−1 , respectively, for low and high doses. Highly dose-related concentrations within subjects, but large variability between subjects were observed. MAMP concentrations were above the 0.2 ng mg−1 cut-off for at least 2 weeks following administration of both low and high doses. The overall AMP/MAMP ratio ranged from 0.07 to 0.37 with a mean value of 0.15 ± 0.07, and a median of 0.13. The percentage of MAMP and AMP removed with the washing procedure decreased with time after administration. A strong correlation was found between area under the curve of MAMP (r2 = 0.90, p = 0.00) and AMP (r2 = 0.94, p = 0.00) concentrations calculated for the 3-week period following administration and the total melanin concentration in hair. Significant correlations were observed also between Cmaxand melanin. Conclusions: This study demonstrated that despite large inter-individual differences, the incorporation of MAMP and AMP into hair is dose-related with much of the observed scatter of MAMP and AMP concentrations explained by melanin concentration in hair