Publisher Summary The oxidation of L-malate in most living organisms is catalyzed by two distinct types of pyridine nucleotide-dependent enzymes. In one case the principal product is oxaloacetate, while in the other it is pyruvate and CO 2 . The enzymes of the malate-oxaloacetate class, which utilize NAD + , have been referred to as simple dehydrogenases, while enzymes of the malate-pyruvate type, which use NADP + , have been designated decarboxylating dehydrogenases and are commonly known as “malic” enzymes. The simple malate dehydrogenases occur in virtually all eukaryotic cells in at least two unique forms identified as mitochondria1 (m-MDH) and soluble or cytoplasmic (s-MDH) according to their cellular location. The soluble isozyme is generally considered to take part in the cytoplasmic side of the malate shuttle, providing a means of transporting NADH equivalents, in the form of malate, across the mitochondrial membrane. The mitochondrial enzyme, in addition to its role in the other half of the malate shuttle, is also a necessary component of the tricarboxylic acid cycle. The microbody malate dehydrogenase found in some plants appears to function in the glyoxylate cycle or possibly in photorespiration. This chapter discusses the NAD + -dependent malate dehydrogenases of animal tissue for which the most structural, mechanistic, and regulatory data are available. Malate dehydrogenase has been identified in a wide variety of sources and purified to homogeneity from a number of them. The chapter focuses on the similarities and differences characterizing the cytoplasmic and mitochondrial enzymes.