The binding of [3H]diazepam and [3H]ethyl-beta-carboline carboxylate (beta-CCE) to rat brain membranes has been studied following injection of the ligand via a tail vein. "Ex vivo" binding was avoided by homogenising the tissue in an excess of unlabelled ligand. The dissociation rate constant for [3H]diazepam and [3H]beta-CCE was approximately 0.46 min-1 at 0 degree C. Displacement of [3H]diazepam by beta-CCE in vivo showed regional variation: the dose of beta-CCE required to inhibit 50% of [3H]diazepam binding in the cerebellum was one quarter of that required in the cortex, hippocampus, or striatum. However, when diazepam was used to displace [3H]beta-CCE in vivo the converse occurred: the dose needed for 50% inhibition in the cerebellum was more than four times that required in the other three regions. These findings support suggestions from in vitro experiments that two receptors exist with different affinities for benzodiazepines and beta-carbolines. The benzodiazepine receptor antagonist Ro 15-1788 did not differentiate between the two receptor subtypes.