One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.