The NADP-dependent isocitrate dehydrogenase of human heart was characterized with respect to molecular size, chemical composition, and catalytic properties. The enzyme appears to exist as a single polypeptide chain with a molecular weight of approximately 53.000, with valine as the N-terminal amino acid. No carbohydrate was detected by staining enzyme on polyacrylamide gels with the periodic acid-Schiff base reagent. The amino acid composition of human heart NADP-specific enzyme is very similar to that of the corresponding enzyme from pig and ox hearts. Statistical analytical methods applied to amino acid compositions of several isocitrate dehydrogenases indicate a strong resemblance between the mammalian heart enzymes but little similarity between the heart enzymes and those isolated from either liver or bacteria. Human heart enzyme cross-reacts with rabbit antibody raised against pig heart NADP-dependent isocitrate dehydrogenase, supporting the suggestion of similarity between these two mammalian enzymes. The apparent Michaelis constant for three- D s -isocitrate was determined to be 2.2 μM for the human heart enzyme. On the basis of the effect of varying the manganous ion concentration on the apparent Michaelis constant for threo- d s -isocitrate, it is postulated that the metal-isocitrate complex is the actual substrate of the enzyme. Citrate, propanetricarboxylate, and threo- l s -isocitrate are weak competitive inhibitors with respect to isocitrate. The essential functional moieties of the coenzyme binding site were examined by testing coenzyme analogs as competitive inhibitors with respect to NADP. the importance of the 2′-phosphata for coenzyme binding was established. The pH dependence of V for the isocitrate dehydrogenase reaction suggests the requirements of a basic form of an essential ionizable group in the enzyme-substrate complex, exhibiting a p K of 5.5, with a heat of ionization of −1.02 kcal/mol. This p K value increases to 5.9 in the presence of 20% ethanol. These results are consistent with the designation of a carboxyl group as the critical ionizable group. Stability studies on the effect of incubating human heart enzyme in normal serum suggest that the absence of isocitrate dehydrogenase activity in serum following myocardial infarction cannot be attributed to instability alone.