Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273 → Thr, Ile-280 → Thr and Gly-317 → Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (∆F102–F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317 → Ser had no effect on respiratory function. However, mutations Met-273 → Thr and Ile-280 → Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.