Translation of the yeast mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) is specifically activated by the products of at least three nuclear genes, PET494, PET54, and PET122. To investigate whether the target site for translational activation is within the 5' untranslated leader of the coxIII mRNA, we asked whether translation of another mitochondrial protein, apo-cytochrome b, from a chimeric mRNA bearing the coxIII mRNA leader required PET494, PET54, or PET122. Mutations in any of these three genes abolished translation of cytochrome b from an mRNA bearing the 5' two-thirds of the coxIII mRNA 5' untranslated leader, showing that all three gene products are required for translation of the chimeric mRNA and must act within the 5' two-thirds of the coxIII mRNA leader. Our data suggest that in wild-type cells, the specific activation of coxIII translation by PET494, PET54, and PET122 occurs by the action of these three gene products at a site or sites in a region of the 5' untranslated leader at least 172 nucleotides upstream of the initiation codon.