By taking advantage of established CD45-deficient DT40 cells, the roles of CD45 in oxidative stress signaling were investigated. Using p-nitrophenyl phosphate as substrate, it was found that CD45 constituted nearly 40% of the total protein-tyrosine phosphatase activity. Almost 90% of the phosphatase activity was rapidly inactivated upon hydrogen peroxide treatment. Hydrogen peroxide-induced tyrosine phosphorylation of cellular proteins and c-Jun N-terminal kinase activation were markedly enhanced in CD45-deficient cells relative to that in its parental cells. In comparison, hydrogen peroxide-induced inositol 1,4,5-trisphosphate production and Ca(2+) mobilization were impaired in CD45-deficient DT40 cells. However, hydrogen peroxide-induced tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2), phosphatidylinositol 3-kinase activity precipitated by anti-phosphotyrosine antibody, and activation of Bruton's tyrosine kinase appeared intact in CD45-deficient DT40 cells. This suggests that CD45 mediates the ability of hydrogen peroxide-activated PLCgamma2 to hydrolyze its substrate via a mechanism independent of both tyrosine phosphorylation of PLCgamma2 and phosphatidylinositol 3-kinase, as well as activation of Bruton's tyrosine kinase. Taken together, our observations demonstrated that, in addition to its negative regulatory or phosphatase activity, CD45 has a positive role in oxidative stress signaling.