The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.