The effects of the enantiomers of the neurosteroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one (DHP), and the benz[e]indene, BI-1, on gamma-aminobutyric acid (GABA) responses were studied using whole-cell recording techniques in cultured rat hippocampal neurons and human embryonic kidney cells (HEK-293) transfected with either alpha 1 beta 2 gamma 2 or alpha 6 beta 2 gamma 2 GABAA receptor subunits. At 10 microM, the (+)-enantiomers enhanced currents gated by 2 microM GABA in all cells, whereas the (-)-enantiomers were significantly less effective. The enhancement of 2 microM GABA responses in HEK-293 cells transfected with alpha 6 beta 2 gamma 2 subunits was about half that of hippocampal neurons or HEK-293 cells transfected with alpha 1 beta 2 gamma 2. The lower sensitivity of alpha 6 beta 2 gamma 2 receptors for (+)-DHP and (+)-BI-1 is accounted for by their greater apparent affinity for GABA. When the GABA concentration was decreased to 0.5 microM to take into account the four-fold higher apparent affinity of alpha 6 beta 2 gamma 2 receptors, these receptors exhibited enhancement similar to alpha 1 beta 2 gamma 2 receptors. These results indicate that both native and recombinant GABAA receptors have enantioselective sites at which neurosteroids and benz[e]indenes modulate GABA responses, and that differences in agonist affinity contribute to apparent differences in steroid sensitivity among GABAA receptors.