The binding of the novel ligand [3H]Ro 5-4864 to membrane preparations of rat kidney and brain was studied. [3H]Ro 5-4864 binds with high affinity (Kd = 0.6 nM) to a single saturable population of benzodiazepine recognition sites on renal membranes. Binding is rapidly reversible and, based on its pharmacological spectrum, takes place at the peripheral-type, Ro 5-4864-sensitive receptor. Specific high-affinity (Kd = 1.1 nM) [3H] Ro 5-4864 binding to the peripheral-type benzodiazepine binding site can also be demonstrated using rat brain membranes. [3H] Ro 5-4864 lacks stereospecificity with regard to chiral activity in position 3. A comparison of benzodiazepine inhibitory potency and structural features reveals that whereas a 4'-substitution assures specificity for the peripheral-type receptor, an N-methyl moiety is essential for optimal activity. [3H]Ro 5-4864 binding to brain membranes is temperature sensitive and is not modulated by barbiturates, convulsants, gamma-aminobutyric acid and chloride anions. The pyrazolopyridine derivative tracazolate inhibits [3H] Ro 5-4864 binding. The regional and subcellular distribution of binding is distinctly different from that previously demonstrated for [3H]benzodiazepine binding in the brain. The olfactory bulb shows the highest binding density, whereas the cerebral cortical, striatal and hippocampal areas are lowest among those areas studied. In the brain, [3H]Ro 5-4864 binding was found to sediment with the nuclear fraction. In conclusion, the present study shows that [3H]Ro 5-4864 is a selective ligand of the peripheral-type benzodiazepine binding site that can unequivocally be demonstrated in the kidney as well as the brain. The physiological significance of these findings, however, remain to be established.