Tolerance occurs to a number of the pharmacodynamic effects of benzodiazepines. To assess pharmacokinetic and neurochemical aspects of tolerance, lorazepam (LRZ) was administered chronically to mice via implantable osmotic pumps and rotarod ataxia, plasma and brain LRZ concentrations, benzodiazepine receptor binding in vivo and in vitro, chloride channel binding and muscimol-stimulated chloride uptake were examined in various brain regions over a 14-day period. Behavioral tolerance, indicated by diminished rotarod ataxia, developed at all doses examined (1, 2, 4 and 10 mg/kg/day), with little change occurring before day 4. The greatest decrease in rotarod ataxia occurred between days 4 and 7. Plasma and brain LRZ concentrations were proportional to dose and were constant over time at each dose, indicating that tolerance was not pharmacokinetic. Benzodiazepine receptor binding as determined by the specific uptake of [3H]Ro15-1788 decreased in cortex, hypothalamus and hippocampus primarily between days 4 and 7, with an approximately 50% decrement in each region by day 7. Receptor binding and rotarod ataxia in cortex were highly correlated at each dose. Apparent affinity in vivo at the receptor was unchanged in cortex, indicating that altered ligand uptake was due to decreased receptor number. Similar results were observed in membrane preparations. There was a small, nonsignificant decrease in chloride channel binding at day 7 compared to day 1. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes was decreased at day 7 compared to day 1. Thus, downregulation of benzodiazepine receptor binding and of gamma-aminobutyric acidA receptor function is closely associated with behavioral tolerance to benzodiazepines.