In isolated hepatocytes the entry into urea of metabolic 1 4 CO 2 ; derived from [ 1 4 C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. 1 4 CO 2 , derived from [1- 1 4 C]alanine enters into urea more readily than 1 4 CO 2 arising from [1- 1 4 C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO 2 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.