This study was aimed to estimate the participation of reactive oxygen species (ROS), other than singlet oxygen (1O2), in the antitumor effect of photodynamic therapy (PDT) with hematoporphyrin derivative (HPD) as well as to determine the ability of photoexcited HPD to the formation of protein peroxides that currently are regarded as a new form of ROS. Studies were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in phosphate-buffered saline and then irradiated with red light at 630 run in the same buffer. Experiments indicated that H2O2 and oxygen radicals could mediate the tumoricidal action of HPD-PDT; we found that photosensitization of EAC cells with HPD leads to the formation of significant amounts of H2O2, superoxide (O2-.), and hydroxyl (OH.) radicals, which along with 1O2 were involved in photoinactivation of the cells in vitro. Our data showed that in EAC cells subjected to HPD-PDT, the generation H2O2, O2-., and OH. could be largely mediated by: (i) an increase in the activity of xanthine oxidase (XOD), due most probably to the conversion of xanthine dehydrogenase (XDH) to XOD via a Ca2+-dependent proteolytic process as well as oxidation of SH groups in XDH; and (ii) photooxidation of some cellular constituents (proteins). Another interesting finding of our studies is that in tumor cells subjected to HPD-PDT the Fenton-like reactions could play an important role in the generation of OH., and that cell-bound Cu/Zn-superoxide dismutase as well as catalase can protect tumor cells against the phototoxic action of HPD. In addition, we clearly demonstrated the ability of photoexcited HPD to the generation of protein peroxides in tumor cells. Studies suggest that 1O2 is the main agent responsible for the generation of protein peroxides in EAC cells treated with HPD-PDT, although other ROS (H2O2, O2-., and OH.) were also implicated in this process. However, further work is needed to clarify the significance of these peroxides in the antitumor effect of PDT with HPD.