The site of localization of TCA cycle dehydrogenases in mitochondria has been investigated by observing the dehydrogenase activities and fine structure of the fractionated samples after freezing and thawing or sonication of beef heart and rat liver mitochondria. 1. In the sonicated mitochondria, activities of malic and isocitric dehydrogenases were highest in the supernatant fraction centrifuged at 198,000 x g for 60 minutes, while the specific activity of a-ketoglutaric dehydrogenase was higher in the fluffy or residue fraction. The distribution of the activity of pyruvic dehydrogenase was similar to that of a-ketoglutaric dehydrogenase. 2. In a sucrose density gradient fractionation of the fluffy fraction obtained by centifugation of sonicated mitochondria at 198, 000 x g for 60 minutes, the activities of malic and pyruvic dehydrogenase were observed in the top (or low density) layer in the form of fine particles, while that of a-ketoglutaric dehydrogenase was observed in the middle (or medium density) layers in the form of aggregates of fine particles and membranous fragments. 3. In the samples fractionated after freezing and thawing of mitochondria, which were considered to be a relatively mild disruption, the specific activity of a-ketoglutaric dehydrogenase was higher in the residue (submitochondria) fraction than that in the supernatant fraction (centrifuged at 144,000 x g, 30 minutes), and the activity of malic dehydrogenase still remained significantly high in the residue fraction. 4. It was deduced that the TCA cycle dehydrogenases could be localized in the matrix of the mitochondria by a loose binding to the inner membrane.