The nucleotide sequence was determined for a 1.5-kilobase genomic fragment containing the mitochondrial malate dehydrogenase gene (MDH1) of Saccharomyces cerevisiae. The open-reading frame encodes a precursor form of the mature enzyme containing an amino-terminal extension of 17 amino acid residues. In vitro translation experiments confirm that the initial translation product of MDH1 is larger than the mature polypeptide. Transcription of MDH1 initiates at several sites from 83 to 97 nucleotides 5' of the translational start site. Alignment of the amino acid sequence for the mature yeast enzyme with those for mammalian mitochondrial and for Escherichia coli malate dehydrogenases reveals polypeptides of very similar sizes with identical amino acids at 54% and 48% of the residue positions, respectively. The amino acid sequences of the yeast and mammalian mitochondrial targeting sequences are similar but less related than the mature polypeptides. The yeast MDH1 gene is shown to reside on chromosome XI.