The allosteric regulation of specific 35S-TBPS binding to the convulsant site on the GABAA receptor/chloride (Cl-) ionophore complex was studied in various brain regions in an attempt to characterize regional heterogeneity of the protein subunits forming the complex. Bicuculline methiodide (BIC), a GABAA antagonist, enhanced binding in cortex (CTX), substantia nigra (SN) and cerebellum (CBL), inhibited binding in inferior colliculus (IC) and did not affect binding in superior colliculus (SC). Similar results were found in CBL and IC using SR-95531, another GABAA antagonist. The levels of endogenous GABA in the different tissue samples could not account for the regional differences in binding. When the functional regulation of these receptors was measured using 36Cl- uptake in microsomes, muscimol-stimulated uptake was completely blocked by BIC in CBL and IC but was not affected by BIC in SC. Additionally, picrotoxin completely blocked muscimol-stimulated uptake in CBL but had no effect in IC or SC. These findings provide a functional basis for regional heterogeneity of GABAA receptor.