A gas chromatographic-mass spectrometric procedure was employed to identify flurazepam and several of its metabolites in the plasma, cerebrospinal fluid (CSF), and brain of cats after an iv injection of flurazepam. Following tissue redistribution, flurazepam was lost from plasma with a mean half-time of 1.4 hr; although usually not detectable in plasma by 24 hr, significant quantities were found in the brain at this time, particularly in the corpus callosum. N1-Hydroxyethyl flurazepam appeared rapidly in plasma following iv flurazepam, peaked at about 40 min, and then declined with a mean half-time of 2.1 hr. N1-Desalkyl flurazepam accumulated in plasma in the first 6 hr after flurazepam was injected, and then declined slowly with a mean half-time of about 50 hr. At 24 hr, corpus callosum concentrations of the N1-desalkyl flurazepam exceeded those of flurazepam by 16- to 63-fold and produced brain/plasma ratios of 6 to 62 in three cats. CSF concentrations of flurazepam and N1-desalkyl flurazepam did not reflect brain concentrations but only the estimated plasma fractions of the unbound drugs. The results suggest that long term central effects of iv flurazepam are mediated to a large extent by the N1-desalkyl flurazepam, in species in which that metabolite accumulates.