The subcellular distribution of malate, 2-oxoglutarate, citrate, aspartate and glutamate and the mitochondrial and cytosolic pH values were determined in perfused livers from fed and fasted rats. The method of fractionation of the freeze-dried tissue in non-aqueous solvents was employed. The following results were obtained: 1) Di- and tricarboxylates are not equally distributed between mitochondria and cytosol. Under the two metabolic conditions studied, the concentrations of citrate and 2-oxoglutarate are higher and those of glutamate and aspartate are lower in the mitochondria than in the cytosol. The distribution of malate varies with the metabolic state. 2) From the distribution of 5,5-dimethyl-2,4-oxazolidinedione it was calculated that the mitochondrial matrix has a more alkaline milieu than the cytosol. The cytosol is also more acidic than the perfusate. The pH difference between mitochondria and cytosol is 0.3 in the fed state and 0.6 in the fasted one, whereas the pH difference between perfusate and cytosol is 0.4 in the fed state and 0.2 in the fasted one. 3) In livers from fed rats, citrate, malate and 2-oxoglutarate appear to be distributed according to the pH gradient across the mitochondrial membrane, whereas in the fasted state, the distribution does not correspond to the pH gradient. Glutamate and aspartate do not follow the pH gradient in either metabolic state. 4) The data indicate that the pH gradient across the mitochondrial membrane does not reflect the membrane potential or the energy state of mitochondria in the intact cell.