Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H(2)O(2) oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.