Abstract In Saccharomyces cerevisiae the mitochondrial gene responsible for the specification of apocytochrome b (cob-box) is believed to consist of both coding and intervening sequences. Mutations in the latter give rise to pleiotropic phenotypes in vivo, lacking not only cytochrome b but also subunit I of cytochrome oxidase, and producing sets of novel polypeptides. The experiments described here have examined 15 different mit− mutants in this region and demonstrate that these results are faithfully reproduced by isolated mitochondria in vitro. This inference also applies to other types of mutational lesions in coding segments of the cob-box gene and of the gene oxi3, responsible for the specification of subunit I.