Withania somnifera (ashwagandha, Indian ginseng) is an immunostimulant herbal medicine used to improve overall health and prevent diseases, particularly in the elderly. However, the mechanisms underlying its immunostimulant effect is poorly understood. To elucidate the mechanism of Withania somnifera, we investigated the effect of a methanolic extract from the root of Withania somnifera (WS) on nitric oxide (NO) production in J774 macrophages. We found that WS (1-256 microg/ml) produced a significant and concentration-dependent increase in NO production, an effect which was abolished by N(G)nitro-L-arginine methyl ester (L-NAME, 3-300 microM), a non-selective inhibitor of NO synthase (NOS), dexamethasone (10 microM), an inhibitor of protein synthesis and N(alpha-p)-tosyl-L-lysine chloromethyl ketone (TLCK, 0.01-10 microM), an inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Dexamethasone did not have any effect on NO production once NOS had been induced (i.e. 12 h after WS). Moreover, western blot analysis showed that WS increased, in a concentration-dependent fashion, inducible NOS protein expression. These results demonstrate that WS may induce the synthesis of inducible NOS expression likely by acting at transcriptional level. The increased NO production by macrophages could account, at least in part, for the immunostimulant properties of Withania somnifera.