Subunit II of cytochrome c oxidase is a mitochondrially encoded protein required for cellular respiration. A respiration-deficient yeast strain has been isolated and shown genetically to carry a mutation in the subunit II structural gene COX2. The respiration-deficient strain produces no subunit II polypeptide and is missing two major transcripts of the subunit II gene. The mutation, first mapped by rho- recombinational rescue to the 5' end of the gene, has been localized precisely, by DNA sequencing, 58 nucleotides upstream of the COX2 ATG initiation codon. The mutant strain carries a single nucleotide change (A to T) relative to the wild type sequence. This mutation occurs in a sequence with substantial homology to a previously identified yeast mitochondrial promoter consensus sequence. These results provide the first in vivo evidence for the importance of the consensus sequence for promoter function.