The effect of alpha-galactosylceramide (alpha-GalCer) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal cells was studied. alpha-GalCer augmented LPS-induced NO production in mouse peritoneal cells, but not in RAW 264.7 macrophage cells. alpha-GalCer augmented NO production, but not tumor necrosis factor (TNF)-alpha production in LPS-stimulated peritoneal cells. Peritoneal cells produced a significant level of interferon (IFN)-gamma in response to alpha-GalCer and anti-IFN-gamma antibody abolished the augmentation of LPS-induced NO production by alpha-GalCer. Moreover, anti-IFN-gamma antibody prevented the enhanced expression of an inducible type of NO synthase mRNA by alpha-GalCer. alpha-GalCer did not augment LPS-induced NO production in peritoneal cells from natural killer T (NKT)-deficient mice. Therefore, it was suggested that alpha-GalCer might augment LPS-induced NO production in peritoneal cells through release of IFN-gamma from NKT cells.