DPN-linked isocitric dehydrogenase has been purified over 700-fold from bovine heart mitochondrial acetone powder. The purified protein exhibits a major component having a sedimentation constant of 10.3 S, and the molecular weight has been estimated to be about 3 or 4 x 10 to the fifth power. The turnover number was calculated to be about 8000 moles of DPNH formed per minute per mole of enzyme. ADP has been found to affect this enzyme in several ways. The nucleotide stabilizes the enzyme under conditions of low ionic strength. ADP also enhances the activity of the enzyme, and this effect has been found to be due to a marked diminution of the Km for isocitrate. In addition, Km for metal ions is also reduced by ADP. At low isocitrate concentrations, such as may exist in mitochondria, the enzyme is virtually dependent of ADP for activity. The activating effect of ADP is highly specific since, of a large number of nucleotides tested, only ADP and dADP are stimulatory. In the presence of low concentrations of isocitrate, the pH optimum is displaced from pH 6.7 in the absence of ADP to about pH 7.2 in the absence if ADP leads to similar shift of the pH optimum. The enzyme is inhibited by DPNH, ATP, and ADPR; the inhibition is competitive with DPN+. TPNH potentiates the DPNH inhibition, and both TPNH and DPNH apparently can form fluorimetrically discernible complexes with the enzyme. On the other hand, the TPN-linked isocitric dehydrogenase of bovine heart has been found to be insensitive to DPN+, DPNH, ATP, and ADP. The mechanism of activation by ADP is probably a conformational change in the enzyme which results in the active site become more accessible to substrate. In the ultracentrifuge, it has been shown that the sedimentation velocity of the enzyme is markedly increased by ADP, a finding which suggests that aggregation has occurred. The significance of these finding has been discussed in terms of a possible positive-plus negative feedback control mechanism for mitochondrial oxidation. In addition, stereospecific synthesis of threo-D-isocitrate-?-T and of threo-Ds-isocitrate-?-T have been accomplished enzymically. Oxidation of these compounds by DPN-linked isocitric dehydrogenase revealed that the ?-hydrogen of isocitrate was transferred stereo-specifically and directly to the ?-side of DPN+, and that the ?-hydrogen of isocitrate was retained in ?-ketoglutarate. The ?-hydrogen was also retained during the reaction with TPN-linked isocitric dehydrogenase, thus indicating that the enol form of oxalosuccinate is not likely to occur as a free intermediate in the oxidation of isocitrate. Thus, in all respects, the hydrogen transfer mediated by DPN-linked isocitric dehydrogenase was the same as that catalyzed by the TPN-specific enzyme.