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The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.