[3H]Ro 15-4513, a partial inverse agonist at the benzodiazepine receptor, binds to two sites in the rat cerebellum, only one of which is sensitive to diazepam. The diazepam-insensitive component, whose identity is unknown, is unique to this brain area. We studied the binding of [3H]Ro 15-4513 in cultured cerebellar granule cells to characterize its binding sites in a specified neuronal cell population and to determine the effects of ethanol on the binding. We also compared the properties of [3H]Ro 15-4513 binding in washed membranes of cultured cells and 14-day-old rat cerebella. [3H]Ro 15-4513 had two binding components in intact granule cells, one sensitive to diazepam, that probably represents binding to the benzodiazepine agonist site, the other sensitive to an antagonist (Ro 15-1788) and two inverse agonists (ethyl-beta-carboline-3-carboxylate, beta CCE and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) of the benzodiazepine receptor (diazepam-insensitive binding). This diazepam-insensitive binding was stimulated by GABA; the maximal increase in binding was about 60% with an EC50 of 0.3 microM. The effect of GABA (10 microM) on the diazepam-insensitive binding was unaffected by 100 microM nipecotic acid but was partially inhibited by 100 microM bicuculline. The stimulation by GABA was also seen at 37 degrees C with washed membranes of 14-day-old rat whole cerebella in the presence of micromolar diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)