The importance of mitochondria for the induction of apoptosis by photodynamic therapy (PDT) was studied with a new photosensitizing dye, methylene blue derivative (MBD), and light. By using fluorescence microscopy and by measuring the MBD-PDT-induced inhibition of specifically subcellularly localized marker enzymes, we show that MBD is localized in mitochondria and not in lysosomes, endoplasmic reticulum or Golgi apparatus of V79 Chinese hamster fibroblasts. Cellular uptake kinetics and fluorescence properties of the dye in cells were characterized. Cell death was studied by a cell survival assay and by flow cytometry of cells stained using the terminal deoxynucleotidyl transferase (TdT) assay. MBD with light induced cell death by apoptosis via 2 different pathways, one rapid and one delayed, depending on the amount of dye in the cells. Cells treated with an MBD concentration higher than 0.05 microg/ml died by apoptosis within 3 hr after light exposure. At a concentration of 0.05 microg/ml MBD, cell death was induced slowly, and apoptotic cells appeared increasingly from the second day after PDT. Combination studies with 2-deoxyglucose (2-DOG) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), inhibitors of glycolysis and oxidative phosphorylation, respectively, indicated that MBD and light inhibited mitochondrial oxidative phosphorylation. Abolishment of both energy sources led to cell death by necrosis within 6 hr. Inhibition of glycolysis alone induced apoptosis between 3 and 6 hr, while inhibition of mitochondrial oxidative phosphorylation alone led to delayed apoptosis within days.