Neuroactive steroids and other positive modulators of GABA(A) receptors showed regional variation in both the efficacy and potency for modulation of [35S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA(A) receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [35S]TBPS binding was sensitive to the presence of the gamma2 subunit and the nature of the alpha subunit (alpha1 beta2 gamma2S > alpha1 beta2, alpha6 beta2, alpha6 beta2 gamma2S, and alpha6 beta2 delta). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.