The region of yeast mitochondrial DNA between 10.7 and 17.9 map units has been characterized by restriction analysis and DNA sequencing. The DNA sequence was obtained from the partially overlapping genomes of the two rho- mutants DS200/A1 and DS302. Two tRNA genes have been found in the sequence upstream of the oxi1 gene. The deduced secondary structures indicate that the genes code for the methionine (5'-CAU-3') and the asparagine (5'-GUU-3') tRNAs of yeast mitochondria. The region between 10.7 and 17.9 units contains two reading frames. One of these corresponds to the oxi1 gene previously shown to code for subunit 2 of cytochrome oxidase (Coruzzi, G., and Tzagoloff, A. (1979) J. Biol. Chem. 254,. 9324-9330; Fox, T. D. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6534-6538). The second reading frame can potentially code for a basic protein with 386 amino acid residues. It is not known at present if this putative gene is translated in vivo. Northern blots of wild type mitochondrial RNA were hybridized to single-stranded probes from the oxi1 gene and flanking regions. The results of these analyses indicate that the primary transcript of the oxi1 region is a high molecular weight RNA (larger than 3 kilobase pairs) which is processed in discrete steps to a mature 850-nucleotide messenger. The 5' leader of the messenger has been established to be 54 nucleotides long and to have a sequence identical with that of the genomic DNA immediately upstream of the oxi1 gene.