The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of a pet strain collection stems from their failure to synthesize cytochrome oxidase. The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme. The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme a biosynthesis. These data suggest that the mutants are likely to be impaired in assembly of the enzyme. A gene designated COX15 has been cloned by transformation of mutants from complementation group G4. This gene is identical to reading frame YER141w on chromosome 5. To facilitate further studies, Cox15p has been expressed as a biotinylated protein. Biotinylated Cox15p fully restores cytochrome oxidase in cox15 mutants, indicating that the carboxyl-terminal sequence with biotin does not affect its function. Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane. The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process. The existence of COX15 homologs in Schizosaccharomyces pombe and Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms.