The effects of enantiomers of the neurosteroid analogues, 3alpha-hydroxy-5alpha-pregnan-20-one (DHP) and 3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile (ACN), and the benz[e]indene, BI-1, on synaptic currents were examined in microcultures of rat hippocampal neurons. Over the range of 0.1-10 microM, the (+)-enantiomers were more potent and effective than their (-)-enantiomeric counterparts in enhancing gamma-aminobutyric acid (GABA)A receptor-mediated evoked synaptic currents. The (+)-enantiomers had small effects on peak currents, but slowed the decay of inhibitory synaptic currents, resulting in 2-3-fold increases in charge transfer during inhibitory synaptic events at 10 microM. Similar prolongations of spontaneous miniature inhibitory postsynaptic currents (IPSCs) and responses to brief GABA pulses to outside-out patches suggest that the prolongations of evoked synaptic currents result primarily from postsynaptic effects. In contrast, the (-)-enantiomers had little effect on evoked IPSCs at concentrations < or = 1 microM, but enhanced inhibitory transmission at 10 microM. At concentrations < or = 1 microM, neither the (+)- nor (-)-enantiomers altered glutamate-mediated excitatory synaptic currents. At 10 microM, (+)-DHP and (+)-ACN depressed excitatory responses in a bicuculline-sensitive fashion, suggesting that direct chloride channel gating by the steroids contributed to the depression. These data indicate that certain steroids and benz[e]indenes augment inhibitory synaptic transmission enantioselectively and provide strong support for the hypothesis that steroids act at specific sites on synaptic GABA(A) receptors rather than via alteration of membrane lipids.