The gene for yeast cytochrome c oxidase subunit V, COX5, has been isolated from a Saccharomyces cerevisiae DNA library by complementation of a cytochrome c oxidase subunit V mutant, JM28. One complementing plasmid, YEp13-511, with a DNA insert of 4.8 kilobase pairs, has been characterized in detail. This plasmid restores respiratory competency in JM28, results in increased cytochrome c oxidase activity and a new form of subunit V in JM28 mitochondria, and is capable of selecting mRNA for subunit V. These results indicate that YEp13-511 carries the COX5 gene and that the subunit V encoded by this plasmid gene is capable of entering the mitochondrion and assembling into a functional holocytochrome c oxidase.