Neuroactive steroids can either potentiate or inhibit a variety of membrane channels. Most studies have suggested that the effects are mediated by specific association of the steroid with the affected channel. However, a recent study of the rho1 (GABA-C) receptor (Mol Pharmacol 66:56-69, 2004) concluded that the actions were consistent with an action of the steroid in the lipid bilayer to alter the lateral pressure profile in the membrane. The enantiomers of an optically active compound are expected to have identical physical properties, including interactions with hydrophobic portions of the cell membrane. We have used two pairs of enantiomers (pregnanolone and ent-pregnanolone, allopregnanolone and ent-allopregnanolone) and show that the ability to potentiate (allopregnanolone) or inhibit (pregnanolone) the rho1 receptor is enantioselective. Therefore, these results strongly suggest that the actions of these neuroactive steroids are mediated by interactions with chiral regions of the target protein, rather than by a change in membrane properties (including lateral pressure).