In this work the protonmotive force (delta p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non aqueous solvents. Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar delta p to that found in rats fed a low fat diet, but when we consider the two components of delta p, we find a significant decrease in mitochondrial/cytosolic pH difference (delta pH(m)) and a significant increase in mitochondrial membrane potential (delta psi(m)) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in delta pH(m) and delta psi(m), which represent the respective driving force for their transport. The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.