Although administered as a short-acting hypnotic for sleeping disorders, flunitrazepam, often in combination with alcohol or other drugs, was one of the most frequently abused benzodiazepines over the last 10 years. It has been reported in cases of driving under the influence, and its use is associated with marked psychomotor impairment. Studies over the last five years have investigated the use of oral fluid as an alternative matrix to blood and urine, especially when non-intrusive and quick sampling procedures are important (e.g., screening for drugs of abuse at the roadside and screening and confirmatory workplace drug testing). In this study, Rohypnol (flunitrazepam) was administered to four healthy volunteers, and oral fluid samples were collected by spitting into a polypropylene tube at fixed times between 0 and 6 h after the intake of a tablet of 1 mg. A specific and very sensitive method was developed, both for flunitrazepam and for its main metabolite 7-aminoflunitrazepam, based on solid-phase extraction of the oral fluid samples, stored at +4 degrees C, and gas chromatographic-mass spectrometric analyses using negative chemical ionization with methane as the ionization gas. The heptadeuterated parent compound and metabolite were used as internal standards. The respective limits of detection and quantitation were 0.05 microg/L and 0.1 microg/L for flunitrazepam, and 0.1 and 0.15 microg/L for 7-aminoflunitrazepam. The parent drug could only be detected when the analyses were performed within 12-24 h after collection of the oral fluid samples or when 2% of NaF was added to the collection tubes. The stability of flunitrazepam in oral fluid was poor, even at +4 degrees C, when no NaF was added to the sample. In any case, concentrations remained below 1 microg/L. The metabolite was detected in slightly higher concentrations, with or without the presence of NaF, reaching a maximum of 1-3 microg/L within 2-4 h after administration. In all cases the drug was detectable, but at extremely low concentrations, for 6 h after intake of a normal dose of Rohypnol and it will be an analytical challenge to come up with a sufficiently sensitive onsite test for low-dose benzodiazepines in oral fluid.