We have mapped the gene for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa in three mammalian species and isolated the human COX6AH gene (HGMW-approved symbol COX6A2). The bovine gene was mapped by somatic cell hybrid mapping panels to bovine chromosome BTA 25 with 94-95% concordance. The mouse gene (Cox6ah) was mapped using an interspecific backcross panel from the cross (C57BL/6J x Mus spretus)F1 x Mus spretus probed with the mouse COX VIa-H cDNA. Cox6ah was located on distal chromosome 7, between D7Mit8 and D7Mit13. From the regions of known gene conservation among these three species, we predicted that human COX6AH would be located on chromosome 16p. We hybridized a human x rodent mapping panel of somatic cell hybrids with the human cDNA to confirm this assignment. These data taken together indicated that the human COX6AH gene is located on the short arm of chromosome 16 and facilitated the isolation of the human gene from a chromosome 16-enriched library. The human COX6AH gene spans about 1 kb and contains three exons and two small introns. The sequences of the proximal 5' flanking regions of COX6AH genes are highly conserved between human, bovine, and rodent.