Groups of male CF-1 mice received 3 and 10 mumol/kg diazepam, lorazepam, and oxazepam intravenously. Between 1 min and 24 h after injection, benzodiazepine concentrations were determined by gas chromatography (GLC) in plasma and in one brain hemisphere; in the other hemisphere, ex vivo benzodiazepine receptor occupancy was measured using 3H-flunitrazepam displacement. Based on GLC data, diazepam entered brain rapidly, and was also cleared rapidly, yielding desmethyldiazepam and oxazepam as metabolites in plasma and brain. However, lorazepam and oxazepam entered brain slowly, with brain:plasma equilibrium achieved at 30-60 min; thereafter, the drugs were eliminated from plasma and brain in parallel. The time course and extent of ex vivo occupancy were highly consistent with GLC data (for diazepam, GLC levels were expressed as the sum of diazepam, desmethyldiazepam, and oxazepam, with metabolite concentrations, normalized for molecular weight and for in vitro benzodiazepine receptor affinity.) Between-method correlations were 0.95 or higher. Thus benzodiazepine receptor occupancy is highly dependent on benzodiazepine concentrations in brain. Differences in the time-course of onset and duration of pharmacologic activity between the highly lipophilic benzodiazepine diazepam and the less lipophilic hydroxylated derivatives lorazepam and oxazepam are largely explained by differences in systemic kinetics and in the rate of uptake into brain.