The benzodiazepine derivative flurazepam (FLZ) is widely used as a hypnotic, but the relative contributions of FLZ and its metabolites desalkylflurazepam (DA-FLZ), hydroxyethylflurazepam (ETOH-FLZ), and flurazepam aldehyde (CHO-FLZ) to overall clinical activity remain uncertain. A single 20 mg/kg dose of FLZ·HCl was administered to mice, with plasma and brain concentrations of FLZ and metabolites determined during 5 h after dosage. Brain and plasma concentrations of FLZ were maximal at 0.5 h after dosage, then declined rapidly in parallel, whereas those of DAFLZ were maximal at 2 h, then declined slowly. Concentrations of ETOH-FLZ, the most polar metabolite, were maximal at 0.5 h, and were undetectable after 3 h. Little CHO-FLZ was detected in either brain or plasma. A single 30-mg oral dose of FLZ·HCl was given to 18 human volunteers, with plasma levels determined over 9 days. FLZ was detected in plasma at low concentrations for no more than 3 h after dosage. ETOH-FLZ concentrations were higher and persisted for 8 h after dosage. CHO-FLZ reached intermediate peak levels and was present longer than FLZ or ETOH-FLZ. In contrast, DA-FLZ achieved the greatest peak concentrations, occurring at 10 h after dosage. Levels declined very slowly, with a mean half-life of 71.4 h, and were still detectable 9 days after FLZ dosage. Plasma free fractions (percent unbound) in mice were 40.3, 51.4, and 25.0% for FLZ, ETOH-FLZ and DA-FLZ, respectively; in humans, values were 17.2, 35.2, and 3.5%, respectively. Brain:free plasma ratios in mice for the three compounds were 8.17, 2.21 and 7.01, and were correlated with HPLC retention times, an index of lipophilicity (r=0.90), suggesting passive distribution from plasma to brain. In vitro specific binding affinities (Ki) in rat brain membranes for FLZ, ETOH-FLZ, DA-FLZ, and CHO-FLZ were 12.7, 16.2, 0.85, and 10.6 nM, respectively. Thus after a single 20 mg/kg dose of FLZ in mice, DA-FLZ brain concentrations greatly exceeded itsKi, while FLZ and ETOH-FLZ levels relative to their ownKivalues are one or more orders of magnitude lower. Since brain:free plasma ratios and binding characteristics for benzodiazepines appear similar in rodents and humans, similar conclusions can be drawn for humans based on pharmacokinetic and protein binding data. Pharmacodynamic effects after a single dose of FLZ in mice and humans are largely attributable to DA-FLZ, consistent with behavioral studies comparing relative potencies of metabolite