In the present study, TONS504 (C51H58N8O5I2; molecular weight, 1,116.9), a novel cationic hydrophilic photosensitizer, was synthesized from protoporphyrin IX dimethyl ester through a five-step process according to a patented method for use in photodynamic therapy (PDT). The subcellular localization of TONS504 and the cytotoxic effects of TONS504-mediated PDT in the mouse mammary tumor EMT6 cell line were investigated. TONS504 was localized primarily in the lysosomes and partially in the mitochondria. The cytotoxic effects of TONS504-mediated PDT in the mouse mammary tumor EMT6 cell line were investigated using a WST8 assay and an Oxidative Stress kit. The cell viability values following treatment with 10 µg/ml TONS504 at light energies of 0, 1, 5 and 10 J/cm2 were 92.5, 101.8, 27.7 and 1.8%, respectively. The percentages of reactive oxygen species (ROS)(+) cells following the same treatment were 8.6, 8.5, 29.2 and 70.1%, respectively, whereas the percentages of apoptotic cells were 7.1, 5.6, 24.8 and 48.7%, respectively. The percentages of ROS(+) and apoptotic cells in the group subjected to TONS504-mediated PDT increased in a manner dependent on the TONS504 concentration and light energy. Further studies are required to evaluate the in vivo pharmacokinetics, tissue distribution and photodynamic effects of TONS504.