gamma-Aminobutyric acid (GABA)-gated Cl- influx was studied in rat brain "microsacs." Midazolam caused a shift to the left of the GABA log dose-response curve. Pentobarbital produced a similar shift plus an increase in maximum response. Diazepam, flurazepam and desalkylflurazepam also enhanced GABA-gated Cl- flux. Their effects were blocked by Ro15-1788, a benzodiazepine antagonist. Acute diazepam pretreatment caused a shift to the left of the GABA dose-response curve but had no effect on the ability of benzodiazepines or pentobarbital to increase GABA-gated Cl- influx. In rats made tolerant by 4 weeks of flurazepam treatment, there was no decrease in the ability of GABA to mediate Cl- flux. GABA was more potent in microsacs from nonwithdrawn rats. In rats withdrawn for 12 but not 48 hr, the maximum GABA response was increased. The ability of benzodiazepines and of pentobarbital to enhance GABA-gated Cl- influx was reduced, showing tolerance. However, 2 days after withdrawal from chronic treatment, this was no longer statistically significant. The results show that benzodiazepine tolerance involves reduced functional coupling between the benzodiazepine recognition site and the GABA recognition site-Cl- channel. Furthermore, reduced effectiveness of GABAA agonists in benzodiazepine-tolerant animals might result from alterations in neuronal activity that occur subsequently to activation of the GABA receptor-gated anion channel.