The promoting effect of hydrogen peroxide (H2O2) against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP⁢) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MPP⁢ resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Addition of H2O2 enhanced the MPP⁢-induced nuclear damage and cell death. Catalase, Carboxy- PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the cytotoxic effect of MPP⁢ in the presence of H2O2. Addition of H2O2 promoted the change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to MPP⁢ in PC12 cells. The results show that the H2O2 treatment promotes the cytotoxicity of MPP⁢ against PC12 cells. H2O2 may enhance the MPP⁢-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that H2O2 as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by neurotoxins.