We have studied the ability of the androgen etiocholanolone and its enantiomer (ent-etiocholanolone) to modulate rat α1β2γ2L GABAA receptor function transiently expressed in human embryonic kidney cells. Studies on steroid enantiomer pairs can yield powerful new information on the pharmacology of steroid interactions with the GABA A receptor. Both steroids enhance currents elicited by GABA, but ent-etiocholanolone is much more powerful than etiocholanolone at producing potentiation. At a low GABA concentration (0.5 μM, <EC5), the presence of 10 μM ent-etiocholanolone potentiates whole-cell currents by almost 30-fold, whereas 10 μM etiocholanolone merely doubles the peak response. At higher GABA concentration (5 μM, ∼EC25), the potentiation curve for ent-etiocholanolone is positioned at lower concentrations than that for etiocholanolone. Single-channel kinetic analysis shows that exposure to etiocholanolone has a single effect on currents: the relative frequency of long openings is increased in the presence of steroid. But exposure to ent-etiocholanolone produces two kinetic effects: an increase in the relative frequency of long openings and a decrease in the frequency of long closed times. The presence of etiocholanolone does not inhibit potentiation by ent-etiocholanolone, suggesting that etiocholanolone is unable to interact with the sites through which ent-etiocholanolone modifies receptor function. The double mutation α1(N407A/Y410F) prevents potentiation by etiocholanolone but not by ent-etiocholanolone, and the α1(Q241A) and α1(I238N) point mutations fully abolish potentiation by etiocholanolone but not by ent-etiocholanolone. We conclude that etiocholanolone and its enantiomer interact with distinct sites on the α1β2γ2L GABAA receptor.