Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF-1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly improved activity over PHP and m-THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m-THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low-level light exposure, and this was charge dependent. The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zinc [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low-level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.