Methylene Blue (MB) has well-established photochemical properties and has been used in a variety of photochemical applications including photodynamic therapy. Despite the fact that most of MB's cytotoxic effects in cells are attributed to mitochondrial damage, the interactions of this dye with mitochondria and the consequent effects on photochemical properties have not yet been fully determined. We monitored MB binding, aggregation and its ability to release singlet oxygen (1O2) on irradiation when interacting with mitochondrial suspensions. MB actively binds to mitochondria and enters the matrix in a manner stimulated by the mitochondrial proton potential and by the increase in mitochondrial concentrations. The greater accumulation of MB in mitochondria with elevated proton potentials or those treated with high concentrations of MB results in the formation of MB dimers, previously shown to be less effective generators of 1O2. Accumulation of MB within mitochondria with high membrane potentials also results in the reduction of MB to the photochemically inactive leuco-MB. Indeed, irradiation of mitochondria with high proton potentials in the presence of MB results in the generation of approximately half the quantity of 1O2 compared with 1O2 generated in mitochondria with low proton potentials. These differences in photochemical properties should influence the cytotoxic effects of photodynamic treatment in the presence of MB.