DNA sequence analysis of mitochondrial genes that code for some mitochondrial proteins has suggested that the opal terminator, UGA, is used as a sense codon in mitochondria. The complete sequences of both the yeast and human genes coding for cytochrome oxidase subunit II contain UGA codons in the reading frame. When the protein sequences predicted by these DNA sequences are compared with the known protein sequence of bovine mitochondrial cytochrome oxidase subunit II, there are regions of homology, in which UGA codons correspond to tryptophan residues. Therefore it has been suggested that UGA specifies tryptophan in the mitochondrial code. We have isolated a yeast mitochondrial tRNATrp and used it to locate the mitochondrial tRNATrp gene in pBR322-mitochondrial DNA recombinants. DNA sequence analysis of this gene revealed that the mitochondrial tRNATrp anticodon is 5'UCA3'. Because there is a U in the wobble position, this tRNA can recognize and insert tryptophan into a growing polypeptide chain in response to the nonsense codon UGA.