1. Mitochondria were isolated from the spongy and compact ventricular myocardium of adult Thunnus thynnus thynnus L. 2. Measurement of the succinoxidase activity of phosphorylating mitochondria revealed that those from the spongy had an apparent Km being half of that of the mitochondria from the compact layer. These latter exhibited Km more sensitive to acidic pH values. 3. Succinate-cytochrome c reductase activity was very differently affected by pH and temperature in non-phosporylating mitochondria from the two cardiac layers. 4. The thermostability of the NADH-cytochrome c reductase and the succinate-cytochrome c reductase activities exhibited also marked differences in the two mitochondrial populations. 5. These results, which represent an example of metabolic zonation at the organelle level, are discussed in relation to the homeostatic cooperation of the two myocardial regions of tuna heart.