Transduction mechanisms associated with ligand binding at diazepam-insensitive subtypes of GABAA receptors remain largely unknown, but unique behavioral effects of ligands binding at these sites have been reported in pigeons. The present study further evaluated the pharmacological characteristics of diazepam-insensitive GABAA receptors in pigeon brain, using [3H]Ro 15-4513. Autoradiography detected diazepam-insensitive benzodiazepine sites on GABAA receptors in a number of brain regions, with the highest densities present in the olfactory bulb, hippocampus, thalamic nuclei and cerebellar granule cell layers, with densities of approximately 10-20% of total benzodiazepine receptor binding. Saturation analysis revealed significant densities (approximately 10% of total benzodiazepine receptor binding) of extracerebellar diazepam-insensitive benzodiazepine receptors in optic lobe, hippocampus, and brainstem compared to 27% in cerebellum. As reported for mammalian diazepam-sensitive benzodiazepine receptors, GABA (50 microM) generally increased the affinities of agonists and partial agonists, had little effect on the affinities of antagonists, and decreased the affinity of an inverse agonist for pigeon cerebellar diazepam-sensitive benzodiazepine receptors. GABA modulation of ligand binding to diazepam-insensitive benzodiazepine receptors was less than that observed for diazepam-sensitive sites, and no positive modulation was observed. These results demonstrate the presence of cerebellar and extracerebellar diazepam-insensitive benzodiazepine receptors in pigeon brain, with distribution patterns and pharmacology similar to those reported in mammals. The comparable central localization and pharmacological properties of drugs at diazepam-sensitive and -insensitive benzodiazepine receptors in pigeons and rats attests to the evolutionary conservation of GABAA systems.