The Sauromatum guttatum alternative oxidase has been expressed in Schizosaccharomyces pombe under the control of the thiamine-repressible nmt1 promoter. Alternative oxidase protein and activity were detected both in spheroplasts and isolated mitochondria, indicating that the enzyme is expressed in a functional form and confers cyanide-resistant respiration to S. pombe, which is sensitive to inhibition by octyl-gallate. Protein import studies revealed that the precursor form of the alternative oxidase protein is efficiently imported into isolated mitochondria and processed to its mature form comparable to that observed with potato mitochondria. Western blot analysis and respiratory studies revealed that the alternative oxidase protein is expressed in the inner mitochondrial membrane in its reduced (active) form. Treatment of mitochondria with diamide and dithiothreitol resulted in interconversion of the reduced and oxidized species and modulation of respiratory activity. The addition of pyruvate did not effect either the respiratory rate or expression of the reduced species of the protein. To our knowledge this is the first time that the alternative oxidase has been effectively targeted to and integrated into the inner mitochondrial membrane of S. pombe, and we conclude that the expression of a single polypeptide is sufficient for alternative oxidase activity.