Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (1O2), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (*O2(-)), hydrogen peroxide (H2O2) and hydroxyl radical (*OH). In this study, we investigated the role of H2O2 in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H2O2 involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT-skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe+2, a co-factor in the Fenton reaction that results in the conversion of H2O2 to *OH. These results provide evidence that formation of H2O2 during irradiation of photosensitized cells contributes to PDT efficacy.