Using 31P NMR and a potassium ion distribution method, the internal phosphorylation potential and the transmembrane electrical potential of intact rat liver mitochondria were measured simultaneously during ATP synthesis and ATP hydrolysis. The ATPase was shown to quickly equilibrate the internal phosphorylation potential with the proton motive force across the mitochondrial membrane. Upon oxygenation of anaerobic mitochondria, there was a very fast increase of delta pH and a quick uptake of inorganic phosphate prior to the buildup of the internal ATP. Consistent with the chemiosmotic theory, these results showed the presence of proton pumping by the respiratory system from the bulk internal aqueous phase to the bulk external aqueous phase. There was, in the measured energetic parameters, no indication of direct energy coupling or short circuiting protons between the respiratory energy input and the ATP synthesis.