Manganese superoxide dismutase (MnSOD) is a nuclear encoded mitochondrial matrix enzyme that scavenges toxic superoxide radicals. It has been shown that increased generation of reactive oxygen species is associated with the differentiation of microorganisms. To test the hypothesis that the ability of mitochondrial superoxide dismutase to neutralize a cellular hyperoxidant state is important for differentiation of mammalian cells, we examined the effect of transfection of MnSOD into mouse embryo fibroblasts on cellular differentiation. C3H10T1/2 cells served as a model for differentiation because these cells can be triggered to differentiate into myoblasts, adipocytes, and chondrocytes by treatment with 5-azacytidine. In this report, myoblast differentiation was defined by the presence of multinucleated cells, appearance of Z-bands, and expression of actin and desmin in the differentiated cells. Transfection of MnSOD gene was found to greatly enhance differentiation of C3H10T1/2 cells into myoblasts by 5-azacytidine. This result identifies MnSOD as an important factor for cell differentiation and supports a role for reactive oxygen species in the process of cellular differentiation.