While IL-18 synergizes with IL-12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL-18 on the Th1/Th2 cytokine response. The injection of alpha-galactosylceramide (alpha-GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN-gamma and IL-4. When the mice were treated 2 h before alpha-GalCer challenge with IL-18, IFN-gamma production but not IL-4 production was remarkably up-regulated. In contrast, pretreatment with IL-18 6 h before the challenge enhanced IL-4 production. However, this IL-18-enhanced IL-4 production was not elicited in mice injected with anti-CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL-18 either 2 h or 6 h before challenge were stimulated with alpha-GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up-regulated in the liver MNC from mice pretreated 6 h before with IL-18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up-regulated the phosphorylation of STAT3 and suppressed in vitro IL-4 production by IL-18-primed liver MNC stimulated with alpha-GalCer, but it did not affect IFN-gamma production. These results suggest that IL-18 time-dependently modulates Th1/Th2 cytokine production in ligand-activated NKT cells by regulating/inducing SOCS3 expression.