The effect of the neurotransmitter gamma-aminobutyric acid (GABA) on high-affinity binding of benzodiazepines to brain membranes has been investigated. GABA stimulated [3H]diazepam binding by more than 100% when extensively washed membranes from brain tissue were used. This GABA-stimulated benzodiazepine binding occurred in all brain regions examined. The stimulation was specific for GABA agonist. It was inhibited by the GABA receptor blocker bicuculline methiodide. A large number of compounds structurally closely related to GABA but without direct effect on the GABA receptor failed to enhance [3H]diazepam binding. The stimulation of benzodiazepine binding was caused by an increase in affinity; the number of binding sites remained unchanged. Half-maximal activation of [3H]diazepam binding occurred in the presence of 300 nM muscimol or 900 nM GABA. beta-Guanidinopropionic acid and imidazoleacetic acid were much weaker activators. It is suggested that the described stimulation of benzodiazepine high-affinity binding is mediated by a receptor for GABA. This site of GABA action exhibits different properties when compared to GABA receptors, as characterized by high-affinity binding of GABA agonists.