In isolated hepatocytes the entry into urea of metabolic 14CO2; derived from [14C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. 14CO2, derived from [1-14C]alanine enters into urea more readily than 14CO2 arising from [1-14C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO2 in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.