The Abl family of mammalian non-receptor tyrosine kinases includes c-Abl and Arg. Recent studies have demonstrated that c-Abl and Arg are activated in the response of cells to oxidative stress. This work demonstrates that catalase, a major effector of the cellular defense against H2O2, interacts with c-Abl and Arg. The results show that H2O2 induced binding of c-Abl and Arg to catalase. The SH3 domains of c-Abl and Arg bound directly to catalase at a P293FNP site. c-Abl and Arg phosphorylated catalase at Tyr231 and Tyr386 in vitro and in the response of cells to H2O2. The functional significance of the interaction is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels. In addition, c-abl-/- arg-/- cells exhibited a marked increase in H2O2-induced apoptosis compared with that found in the absence of either kinase. These findings indicate that c-Abl and Arg regulate catalase and that this signaling pathway is of importance to apoptosis in the oxidative stress response.