Clozapine, an atypical neuroleptic, functionally antagonizes the γ-aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, γ-aminobutyric acid type A (GABA) receptor, in brain vesicles. GABA antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [S] t-butylbicyclophosphorothionate ([S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABA antagonist 2′-(3′-carboxy-2′,3′-propyl)-3-amino-6- p-methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [H]muscimol and [H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant α6β2γ2 receptors, the predominant α6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 μM. In contrast, recombinant α1β2γ2 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant α6β2γ2 and α6β3γ2 receptors resulted in clozapine-insensitive receptors, whereas α6β1γ2 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in α1βxγ2 receptors decreased in the order of α1β1γ2>α1β2γ2>α1β3γ2. The results indicate that clozapine antagonizes the function of most GABA receptor subtypes, and that the interaction is determined by the interaction of the α and β subunit variants. GABA antagonism is a unique property of clozapine, not shared by haloperidol, which might be involved in the pharmacological mechanism for the increased seizure susceptibility associated with clozapine treatment. © 1995, Springer-Verlag. All rights reserved.