An iron-sulfur protein of Complex III was purified by phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography. The purified preparation was homogeneous as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a specific antibody directed against this protein was raised in a rabbit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunoperoxidase reaction indicated that Complex III possesses a single polypeptide which reacts with the antibody. It was also found that the iron-sulfur center-containing subunits identified so far in Complex I did not cross-react with the antibody, indicating that they are antigenically unrelated to the iron-sulfur protein of Complex III.