Pyropheophorbide-a methyl ester (MPPa) is a semisynthetic photosensitizer derived from chlorophyll a. The absorption peak of MPPa in organic solvent and in cells was at 667 and 674 nm, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that MPPa had no dark cytotoxicity. In vitro photodynamic activity was extensively evaluated using a human lung carcinoma cancer cell line (NCI-h446). MPPa exhibited no genotoxicity, as assayed by single-cell gel electrophoresis. Using confocal laser scanning microscopy and organelle-specific fluorescent probes, MPPa was found to localize in the intracellular membrane system, namely the endoplasmic reticulum, Golgi apparatus, lysosomes and mitochondria, in the NCI-h446 cells. Furthermore, nuclear staining and DNA gel electrophoresis revealed that DNA condensation and fragmentation occurred post–photodynamic therapy, indicating the cell death was in the apoptotic mode.