Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-ginsenoside and 20(S)-ginsenoside are epimers. We have shown previously that the mixture of 20(R)- and 20(S)-ginsenosides regulates ion channel activity. However, it was not clear which epimer was responsible. We investigated the structure-activity relationship of the ginsenoside Rg3 stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(R)-Rg3) and 20-S-proto-panaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyr-anoside], (20(S)-Rg3) in regulating voltage-dependent Ca2+, K+ or Na+ channel currents and 5-HT3A and a3b4 nicotinic acetylcholine (nACh) receptor channel currents expressed in Xenopus oocytes. 20(S)-Rg3 but not 20(R)-Rg3 inhibited the Ca2+, K+ and Na+ channel currents in a dose- and voltage-dependent manner. The fact that only 20(S)-Rg3 is active indicates that its hydroxyl group may be geometrically better aligned with the hydroxyl acceptor group in the ion channels than that of 20(R)-Rg3. However, both Rg3 stereoisomers inhibited 5-HT3A and a3beta4 nACh receptor channel currents. These results indicate that the selectivity of action of the Rg3 stereoisomers differs between voltage-dependent and ligand-gated ion channels.