The binding of [3H]Ro 5-4864 to the peripheral-type benzodiazepine binding site in brain is characterized. The binding is saturable, high-affinity (KD = 1.6 nM), and reversible. The comparison of [3H]Ro 5-4864 and [3H]diazepam binding sites reveals major differences which include the following. There are about one-fourth as many peripheral-type binding sites than central sites in brain. Peripheral sites are present in many extranervous tissues and have a brain regional distribution distinct from that of the central-type receptor. The [3H]Ro 5-4864 binding site also is apparently highly localized in the nuclear membrane in contrast to the central-type receptor, which is synaptosomal. gamma-Aminobutyric acid has no effect on [3H]Ro 5-8464 binding, again in contrast to its marked effect on [3H]diazepam binding. Various putative benzodiazepine receptor ligands, such as purines, beta-carbolines, and kynurenamines, are also inactive as inhibitors of [3H]Ro 5-4864 binding. Blocking the benzodiazepine receptor by photoaffinity labeling decreases [3H]diazepam binding by more than 80% and has no effect on [3H]Ro 5-4864 binding. These results indicate that the peripheral-type benzodiazepine binding site in brain is a separate entity whose physiological function is probably distinct from that of the central-type benzodiazepine receptor.