Abstract The malate dehydrogenases of the mitochondria of ox heart, rat liver and rabbit kidney and those extracted from acetone-dried powders of horse, pig and pigeon hearts have been purified and compared by a number of criteria. In substrate and coenzyme specificities, in the values for some Michaelis constants, and in their behaviour during purification, the six enzymes showed a close similarity. The malate dehydrogenases of horse and pig heart are enzymes with a low content of aromatic amino acids, and with alanine as the only N-terminus. Sedimentation and quantitative end-group data for these two enzymes are consistent with molecular weights of 50000–60000.