the oxi3 locus of yeast mitochondrial DNA has been sequenced in Saccharomyces cerevisiae D273-10B. The sequence was obtained from the mitochondrial genomes of a series of cytoplasmic "petite" mutants selected for the retention of genetic markers in the oxi3 locus. The oxi3 locus has been ascertained to code for Subunit 1 of cytochrome oxidase. The Subunit 1 gene is 9,979 nucleotides long, consisting of seven to eight exons that account for only 16% of the gene sequence. The coding sequences have been identified on the basis of protein sequence homology with Subunit 1 of human cytochrome oxidase. The yeast Subunit 1 is 510 amino acid residues long and has a molecular weight of 56,000. In addition to the exon sequences, the Subunit I gene contains six to seven introns. The first four introns have long reading frames that are continuous with the exon coding sequences. These reading frames are potentially capable of coding for basic proteins with molecular weights ranging from 30,000 to 80,000. The first two introns of the gene have a sequence homology of 50%, while the reading frame of the fourth intron is 70% homologous with an intron of the apocytochrome b gene. At least five stable transcripts have been found by Northern blot hybridizations with single-stranded DNA probes containing either exon or intron sequences. A 1.9-kolobase transcript hybridizes only with probes from the exon regions of the gene. This RNA species has been tentatively identified as the fully processed messenger of Subunit 1. Other transcripts are detected with intron probes. Three transcripts with sizes of 2.5, 2.4, and 0.85 kilobases appear to be stable excision products from the first, second, and fifth introns.