The steroid pregnenolone sulfate (PS) interacts with the GABAA receptor complex in a mixed GABA-agonistic/antagonistic manner in binding experiments. However, in functional assays pregnenolone sulfate (at micromolar concentrations) behaves as an allosteric GABAA receptor antagonist, similar to the convulsant, picrotoxin. In the present work, we examined the binding of [3H] pregnenolone sulfate to membranes from rat brain. We report that this steroid binds to two or three populations of sites: (Kd1 300-500 nM, Kd2 about 20 microM and Kd3 about 200-300 microM. [3H]Pregnenolone sulfate binding is thermostable and resistant to protease digestion. Picrotoxin inhibits about 40% of 5 nM [3H]pregnenolone sulfate binding, but other GABA receptor ligands are inactive. The data suggest that [3H]pregnenolone sulfate binding sites are connected with or adjacent to the ionic channel of the GABAA receptor, but that they differ from picrotoxin recognition sites. The high and intermediate affinity pregnenolone sulfate binding sites may mediate GABA-agonistic and antagonistic actions of pregnenolone sulfate, respectively.