Recently we demonstrated that [3H]dehydroepiandrosterone sulfate binds specifically to two populations of sites in rat brain membranes [Majewska et al. (1990) Eur. J. Pharmac. 189, 307-315]. As an extension of this work, we studied the biochemical and pharmacological properties of [3H]dehydroepiandrosterone sulfate binding to brain membranes and the effects of dehydroepiandrosterone sulfate on GABA-induced currents in cultured neurons. [3H]Dehydroepiandrosterone sulfate binding depended upon incubation time, pH, protein concentration, and incubation temperature. Thermal denaturation or pretreatment of the membranes with protease or phospholipase A2 reduced the binding by 54-85%. The higher affinity [3H]dehydroepiandrosterone sulfate binding sites appeared to be associated with protein and with the GABAA receptor complex. Among substances known to interact with the GABAA receptor complex, pregnenolone sulfate, pentobarbital, and phenobarbital inhibited the binding of [3H]dehydroepiandrosterone sulfate. High micromolar concentrations of dehydroepiandrosterone sulfate inhibited [3H]muscimol and [3H]flunitrazepam binding to rat brain membranes, primarily by reducing the binding affinities. Dehydroepiandrosterone sulfate also produced a concentration-dependent block of GABA-induced currents in cultured neurons from ventral mesencephalon (IC50 = 13 +/- 3 microM). The results of this study are consistent with an action of dehydroepiandrosterone sulfate as a negative noncompetitive modulator of the GABAA receptor. Because concentrations of dehydroepiandrosterone sulfate in the brain undergo physiological variations, this neurosteroid may play a vital role in regulation of neuronal excitability in the central nervous system.