A cDNA library was constructed from poly(A)-rich RNA of H35 rat hepatoma cells by insertion into lambda gt11. Screening with an antiserum to rat liver holocytochrome-c oxidase yielded fifteen different recombinant clones. Eight clones were identified using monospecific antisera to individual subunits of the rat liver enzyme. The cDNA clones coding for subunits VIc and VIII were further characterized by DNA sequence analysis after subcloning in pUC8 and M13 mp9. The deduced amino acid sequences show 80% and 60% homology to the corresponding bovine heart subunits, respectively. From the nucleotide sequences we can conclude that subunit VIc is not synthesized as a larger precursor molecule like most other mitochondrial proteins. The size of the mRNAs coding for subunits VIc and VIII is about 450 nucleotides, as revealed by Northern blot analysis with RNAs from different tissues. The clones were further used as probes for Southern blotting. Restricted high-molecular-mass DNA showed a complex pattern of bands indicating multigene families for both subunits in the rat genome.