The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.