In Saccharomyces cerevisiae, COX5a and COX5b encode two distinct forms of cytochrome c oxidase subunit V, Va and Vb, respectively. To determine the relative contribution of COX5a and COX5b to cytochrome c oxidase function, we have disrupted each gene. Cytochrome c oxidase activity levels and respiration rates of strains carrying null alleles of COX5a or COX5b or both indicate that some form of subunit V is required for cytochrome c oxidase function and that COX5a is much more effective than COX5b in providing this function. Wild-type respiration is supported by a single copy of either COX5a or COX5ab (a constructed chimeric gene sharing 5' sequences with COX5a). In contrast, multiple copies of COX5b or COX5ba (a chimeric gene with 5' sequences from COX5b) are required to support wild-type respiration. These results suggest that the decreased effectiveness of COX5b is due to inefficiency in gene expression rather than to any deficiency in the gene product, Vb. This conclusion is supported by two observations: (i) a COX5a-lacZ fusion gene produces more beta-galactosidase than a COX5b-lacZ fusion gene, and (ii) the COX5a transcript is significantly more abundant than the COX5b transcript or the COXsba transcript. We conclude that COX5a is expressed more efficiently than COX5b and that, although mature subunits Va and Vb are only 67% homologous, they do not differ significantly in their ability to assemble and function as subunits of the holoenzyme.