Addition of insulin or a physiological ratio of ketone bodies to buffer with 10 mM glucose increased efficiency (hydraulic work/energy from O2 consumed) of working rat heart by 25%, and the two in combination increased efficiency by 36%. These additions increased the content of acetyl CoA by 9‐ to 18‐fold, increased the contents of metabolites of the first third of the tricarboxylic acid (TCA) cycle 2‐ to 5‐fold, and decreased succinate, axaloacetate, and aspartate 2‐ to 3‐fold. Suc‐ cinyl CoA, fumarate, and malate were essentially unchanged. The changes in content of TCA metabolites resulted from a reduction of the free mitochondrial NAD couple by 2‐ to 10‐fold and oxidation of the mitochondrial coenzyme Q couple by 2‐ to 4‐fold. Cytosolic pH, measured using 31P‐NMR spectra, was invariant at about 7.0. The total intracellular bicarbonate indicated an increase in mitochondrial pH from 7.1 with glucose to 7.2, 7.5, and 7.4 with insulin, ketones, and the combination, respectively. The decrease in Eh⁷ of the mitochondrial NAD couple, Eh⁷NAD∗/NADH, from ‐280 to ‐300 mV and the increase in Eh⁷ of the coenzyme Q couple, Eh⁷Q/QH2, from ‐4 to +12 mV was equivalent to an increase from ‐53 kJ to ‐60 kJ/2 mol e in the reaction catalyzed by the mitochondrial NADH dehydrogenase multienzyme com‐plex (EC 1.6.5.3). The increase in the redox energy of the mitochondrial cofactor couples paralleled the increase in the free energy of cytosolic ATP hydrolysis, ΔGatp‐ The potential of the mitochondrial relative to the cytosolic phases, Emito/cyto, calculated from ΔGatp and ΔpH on the assumption of a 4 H⁺ transfer for each ATP synthesized, was ‐143 mV during perfusion with glucose or glucose plus insulin, and decreased to ‐120 mV on addition of ketones. Viewed in this light, the moderate ketosis characteristic of prolonged fasting or type II diabetes appears to be an elegant compensation for the defects in mitochondrial energy transduction associated with acute insulin deficiency or mitochondrial senescence.—Sato, K., Kashiwaya, Y., Keon, C. A., Tsuchiya, N., King, M. T., Radda, G. K., Chance, B., Clarke, K., Veech, R. L. Insulin, ketone bodies, and mitochondrial energy transduction. FASEB J. 9, 651‐658 (1995)