To validate information on flunitrazepam use, we investigated human hair for flunitrazepam and its major metabolite 7-amino-flunitrazepam by gas chromatography coupled to mass spectrometry in negative chemical ionization mode of detection. Samples were twice decontaminated with methylene chloride, pulverized in a ball mill and 50 mg of powdered hair were incubated in Soerensen buffer (pH 7.6) in the presence of diazepam-d5 used as internal standard. After liquid-liquid extraction of the incubation medium with diethylether-chloroform (80:20, by vol.), the organic phase was evaporated and the dry extract was derivatizated with heptafluorobutyric anhydride. Benzodiazepines were separated on a 30 m capillary column and detected using single ion monitoring. Among 40 hair samples tested (obtained from drug addicts deceased by heroin overdose), 14 were positive for both flunitrazepam and 7-amino-flunitrazepam and 12 for 7-amino-flunitrazepam only. Concentrations ranged from 31 to 129 pg/mg (mean: 60 pg/mg) and from 3 to 161 pg/mg (mean: 46 pg/mg) for flunitrazepam (14 cases) and 7-amino-flunitrazepam (26 cases), respectively. This first report described the detection of flunitrazepam and 7-amino-flunitrazepam in hair of chronic abusers. Due to the low concentrations observed, negative chemical ionization appears to be the alternative to test flunitrazepam and other benzodiazepines in hair.