Mitochondrial malate dehydrogenase (l-ma-late:NAD+ oxidoreductase, EC 1.1.1.37) has been purified from both rat liver and rat heart by using Sepharose-Blue Dextran pseudoaffinity chromatography. Under the conditions employed most of the mitochondrial malate dehydrogenase and some cytosolic malate dehydrogenase are adsorbed, whereas many proteins simply pass through. The blue column is then subjected to several treatments which elute a variety of other proteins (notably residual cytosolic malate dehydrogenase and lactate dehydrogenase) which also have an affinity for the bound dye Cibacron Blue F3GA. Mitochondrial malate dehydrogenase is then eluted by forming an abortive ternary complex with reduced nicotinamide adenine dinucleotide-sodium d(+)-malate. Pooled active fractions are passed through a preequilibrated diethylaminoethyl-Sephadex column which retains contaminating proteins. The active fractions are concentrated, and residual contaminants are removed by very small stepwise pH changes on a CM-52 cellulose column. The procedure is mild and rapid and yields enzyme which is homogeneous by the applied criteria. In addition, lactate dehydrogenase may be copurified. The purified mitochondrial malate dehydrogenases from liver and heart have been characterized and compared. Both enzymes show evidence of multiple forms upon starch or polyacrylamide gel electrophoresis or upon isoelectric focusing. These forms are not generated artifactually during purification since they correspond to forms seen in crude extracts. The heart enzyme contains more high pI forms than the liver enzyme. Sodium dodecyl sulfate electrophoresis indicated identical subunit molecular weights of 35 000 for enzyme from both sources. Ultraviolet spectra were practically identical. The mild isolation procedure produces mitochondrial malate dehydrogenase of high (if not maximal) specific catalytic activity. Amino acid analyses of heart and liver mitochondrial malate dehydrogenase showed almost identical values, and the high levels of amide nitrogen are consistent with the high pi values (9.0-9.5) obtained by column electrofocusing. In addition, the procedure described produces enzymes with small, somewhat variable amounts of tightly complexed glycero-phospholipids. © 1979, American Chemical Society. All rights reserved.