N3-Phenacyluridine (3-phenacyl-1-beta-D-ribofuranosyluracil) has potent sedative and hypnotic activities following intracerebroventricular injection in mice. To study the mechanism of action of N3-phenacyluridine, the interaction of this compound with the benzodiazepine receptor has been investigated. Results obtained showed that this compound inhibited specific binding of [3H]flunitrazepam to synaptic membranes of bovine cortex in a concentration-dependent fashion (IC50 = 129 microM). Scatchard analysis of [3H]flunitrazepam binding revealed that N3-phenacyluridine interacted with the ligand at the benzodiazepine receptor binding site in a competitive manner. Ro15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a ]1, 4-benzodiazepine), a benzodiazepine receptor antagonist, also inhibited the specific binding of [3H]flunitrazepam in the presence of the compound. The results suggest that the pharmacological activity of N3-phenacyluridine may be partially mediated through the benzodiazepine receptor.