GABA-stimulated 36Cl- influx was used to investigate regional differences in response to chronic diazepam treatment by comparing cortical and cerebellar tissue from rats chronically treated with diazepam for 3 weeks. Using a treatment protocol which has previously been shown to produce behavioral tolerance and physical dependence, cortical membrane preparations from chronic diazepam-treated rats were found to exhibit a decreased responsiveness to the stimulation of 36Cl- influx by GABA and a corresponding decrease in the ability of flunitrazepam to enhance GABA-stimulated 36Cl-influx. This decrease in sensitivity to flunitrazepam, however, appears to reflect the underlying decrease in sensitivity to GABA. In contrast, in membrane vesicles prepared from cerebella of chronic diazepam-treated rats, there was no measurable effect on GABA-stimulated 36Cl--influx or on the enhancement of GABA-stimulated 36Cl- influx by flunitrazepam. These results support the suggestion that there is a regionally specific reduction in GABA/benzodiazepine receptor function following chronic benzodiazepine treatment.