We previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin-12 (IL-12) act as anti-tumour/anti-metastatic effectors. However, IL-12 reportedly induces a rapid disappearance of liver NKT cells by activation-induced apoptosis. In the present study, however, we show that injection of IL-12 into mice merely down-regulates the NK1.1 expression of liver NKT cells and Vbeta8+ intermediate T-cell receptor cells and CD1d/alpha-galactosylceramide (alpha-GalCer)-tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL-12-pretreated (24 hr before) mice were injected with alpha-GalCer, not only serum interferon-gamma but also serum IL-4 concentrations increased several-fold in comparison to the control alpha-GalCer-injected mice. However, IL-12 pretreatment markedly up-regulated serum ALT levels and Fas-ligand expression on NKT cells after alpha-GalCer injection in middle-aged mice only. Consistently, the liver mononuclear cells (MNC) from IL-12-pretreated mice stimulated with alpha-GalCer in vitro produced much greater amounts of interferon-gamma and IL-4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate-buffered saline. Liver MNC from middle-aged mice, but not from young mice pretreated with IL-12, also showed increased cytotoxicity following in vitro alpha-GalCer stimulation against cultured hepatocytes. Furthermore, IL-12 treatment of middle-aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vbeta8+ T cells, and in vitro experiments also revealed that IL-12 pretreatment of liver MNC from middle-aged mice enhanced their tumour necrosis factor-alpha production after alpha-GalCer stimulation. Synthetic ligand-mediated functions of NKT cells, including IL-4 production, are thus enhanced by IL-12 pretreatment.