Extracts from yeast cells grown under different conditions and from mitochondria were subjected to agar gel electrophoresis. Three bands showing malate dehydrogenase activity were found in extracts from aerobic derepressed cells (c‐MDH I, c‐MDH II, and m‐MDH III), whereas generally only one band was present in extracts from anaerobic repressed cells (c‐MDH I) and another one in extracts from mitochondria (m‐MDH III) of aerobic derepressed cells.The different malate dehydrogenases showed dissimilar regulatory and physical properties, e. g. sensitivity to inactivation by heat, inhibition by oxaloacetate and by p‐chloromercuribenzoate, activation by Pi, Michaelis constants etc. A separation of malate dehydrogenases by DEAE‐cellulose chromatography was performed giving only two malate dehydrogenase forms. These two forms were subjected to agar gel electrophoresis and gave one band each, corresponding to the cytoplasmic c‐MDH I and c‐MDH II. The possible metabolic significance of the three malate dehydrogenase isoenzymes is discussed.