The unnatural enantiomers of dehydroepiandrosterone sulfate (1), pregnenolone sulfate (2), and (3α,5β)-3-hydroxypregnan-20-one sulfate (3), compounds 4-6, respectively, were prepared by total steroid synthesis. The enantioselectivity of the compounds as negative modulators of the GABA(A) receptors present in cultured rat hippocampal neurons was examined using electrophysiological methods. Enantioselectivity was found for the inhibitory actions of the dehydroepiandrosterone enantiomers. The IC50s for compounds 1 and 4 were 11 ± 1 and 80 ± 14 μM, respectively. Little, if any, enantioselectivity was found for the other two pairs of steroid sulfate inhibitors. The IC50s for compounds 2 and 5 were 82 ± 12 and 76 ± 27 μM, respectively. The IC50s for compounds 3 and 6 were 39 ± 7 and 46 ± 2 μM, respectively. The results suggest that the sites of action for the androstane and pregnane series of steroid sulfate blockers of GABA-mediated current are different. The observed enantioselectivity for the actions of dehydroepiandrosterone sulfate indicates that its inhibitory actions are mediated via a chiral recognition site and provides new evidence in support of the earlier hypothesis that there is a binding site for this compound on GABA(A) receptors. Conversely, the failure to observe enantioselectivity for the actions of pregnenolone sulfate and steroid sulfate 3 indicates that a chiral recognition site for these steroids does not exist on GABA(A) receptors and suggests that the effects of these compounds on this receptor's function may arise indirectly as a consequence of steroid-induced membrane perturbation.