Barbiturates have been reported to augment benzodiazepine receptor affinity in vitro, but their effects in vivo are uncertain. We determined benzodiazepine receptor binding in vivo by specific uptake of [3H]Ro15-1788 after barbiturate administration. Pentobarbital (30 mg/kg) increased receptor binding in cerebral cortex and cerebellum at 30 min after injection, with a peak effect occurring at 1 h after dosage, and a return to control levels at 2 h. Specific binding was increased at 1 h after pentobarbital administration in a dose-dependent fashion (7.5-90 mg/kg). Pentobarbital at doses up to 30 mg/kg failed to alter nonspecific binding, but at doses of 60 mg/kg increases in nonspecific binding were observed. The increases in specific binding observed after barbiturate administration were most likely due to a change in apparent receptor affinity, as determined by administration of varying doses of clonazepam to pentobarbital-treated (30 mg/kg) animals. The order of potency of a series of barbiturates in augmenting benzodiazepine receptor binding in cerebral cortex and cerebellum in vivo was: secobarbital greater than pentobarbital greater than amobarbital greater than phenobarbital greater than barbital. The same relative rank order of potency exists for the anesthetic/hypnotic activity of these barbiturates. These data suggest that barbiturates increase the apparent affinity of benzodiazepine receptors in vivo; unlike their in vitro actions, these alterations can be detected with a receptor antagonist.