We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.