The effect of H2O2 and catalase on isolated rat cerebellum nitric oxide (NO) synthase activity was determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline. H2O2 (1-5 mM) markedly increased NO synthase activity in the presence of endogenous catalase (72 +/- 4 U/mL). This effect of H2O2 was further increased by exogenous catalase (200 U/mL). Exogenous catalase (0.1 to 1000 U/mL) by itself had no significant effect on NO synthase activity. Nitroblue tetrazolium chloride, an electron acceptor, inhibited NO synthase activity in a concentration-dependent manner. This study suggests that H2O2 is not directly involved in NO synthesis and that the H2O2/catalase stimulation of NO synthase activity may be due to the excess oxygen produced by the H2O2/catalase system.