Steroids that enhance gamma-aminobutyric acid (GABA)A receptor function in the central nervous system allosterically modulate the binding of the convulsant chloride channel ligand [35S]-t-butyl bicyclophosphorothionate. When assayed in membrane homogenates and in tissue sections by autoradiography, concentration-dependence curves vary with respect to both brain region and the nature of the steroid. Alphaxalone and endogenous steroid hormone metabolites inhibit the binding of [35S]-t-butyl bicyclophosphorothionate in some regions, enhance it in others and give biphasic concentration-dependence in others, apparently the result of algebraic summation of two effects involving regional-dependent enhancement or inhibition. The alphaxalone effect is additive with that produced by adding GABA to the binding assays in some regions, but synergistic in other areas. Likewise, the effect of GABA is inhibited completely by saturating concentrations of the antagonist bicuculline methochloride in some areas but only partially in others, and completely or partially reversed by the convulsant benzodiazepine Ro5-4864, depending on region. The granule cell and molecular layers of cerebellum are particularly different in these allosteric interactions. The heterogeneity of binding behavior is consistent with the presence of multiple GABAA receptor subtypes in the brain. Regional variation in subunit gene expression apparently produces a family of hetero-oligomeric GABAA receptors with different biological and pharmacological properties, including qualitative and quantitative differences in modulation by neuroactive steroids.