Neurosteroids directly modulate ligand gated ion channels such as GABA_A receptors. Two such molecules, 3b-OH A-ring reduced pregnane steroids and pregnenolone sulfate (PS), inhibit recombinant GABA-A receptor. Using a two-electrode voltage-clamp technique, we compared the effect of 5a-pregnan- 3b,20(S)-diol (UC 1019), 5b- pregnan-3b,20(R)-diol (UC1020) and PS on the activation onset and offset time of the recombinant GABA_A receptor (rat a1b2g2L) in Xenopus oocytes. Rapid solution changes allowed the kinetic analysis of GABA-evoked currents. Steroids were co-applied with 30 mM GABA for 10 s, followed by a 80 s wash out period. PS (>=0.3 mM) moderately increased the slow onset rate (k_on-S) of GABA-response. PS had no significant effects on the fast onset rate (k_on-F). UC1019 and UC1020 decreased the k_on-S of the GABA-response in a concentration-dependent manner with no significant effects on the k_on-F. Like PS, UC1019 and UC1020 decreased the slow offset rates (k_off-S). In addition, PS increased the fast offset rate (k_{off- F}) in a concentration-dependent manner, while UC1019 and UC1020 decreased k_{off- F}. The EC50 of PS to increase k_off-F was calculated as 0.470.1 mM. The corresponding IC50 values of UC1019 and UC1020 to decrease k_off-F were 5.00.5 mM and 8.40.9 mM, respectively. These results suggest differential actions of PS and 3b,20(R/S)-pregnandiols on the offset time course of GABA-site activation.