Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of brain benzodiazepine receptors, has been shown to antagonize some actions of ethanol. In addition to conventional benzodiazepine binding sites, Ro 15-4513 binds to a specific cerebellar protein, the binding of which has been shown to be insensitive to diazepam. The binding of [3H]Ro 15-4513 was studied in washed membranes of the cerebellum, hippocampus, and cerebral cortex of two rat lines developed for differences in their sensitivity to ethanol-induced motor impairment. Only minor differences were found in the estimated parameters (KD and Bmax) for the total specific binding between the rat lines. The main difference between the rat lines was, however, observed in the characteristics of the cerebellar binding, all of which was displaced by diazepam in most of the alcohol-sensitive [alcohol-nontolerant (ANT)] rats, in contrast to only approximately 75% displacement in most of the alcohol-insensitive [alcohol-tolerant (AT)] ones. The following cerebellar results were obtained with the major subgroups of both lines, i.e., with the AT rats chosen for the presence of the diazepam-insensitive binding and with the ANT rats chosen for its absence. The KD for the total specific [3H]Ro 15-4513 binding in the ANT animals was about half of that in the AT animals. No line difference was found in the Bmax of the binding in these rats. Photolabeling with [3H]Ro 15-4513 showed that the diazepam-insensitive binding was in a protein with a molecular weight of 55,000.(ABSTRACT TRUNCATED AT 250 WORDS)