The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.