The purpose of this study was to find whether the concentration of N-acetylglutamate and ornithine transport into mitochondria would regulate urea synthesis when the dietary protein quality was manipulated. Experiments were done on three groups of rats given diets containing 10 g of gluten, 10 g of casein, or 10 g of whole egg protein/100 g for 10 days. The plasma concentration and urinary excretion of urea, the liver concentration and synthesis of N-acetylglutamate, the liver concentrations of glutamate and lysine, and the liver ornithine transport into mitochondria increased with the decrease in quality of dietary protein. A reverse correlation was observed between the activities of urea cycle enzymes, the plasma concentration of arginine, and urinary excretion of urea under these conditions. N-Acetylglutamate concentration and ornithine transport into mitochondria in the liver were closely correlated with the excretion of urea. These results suggest that greater N-acetylglutamate concentration and ornithine transport into isolated mitochondria in the liver of rats, given the lower quality of protein, stimulate urea synthesis and that the concentrations of glutamate and lysine in the liver are at least partly related to the hepatic N-acetylglutamate synthesis and ornithine transport, respectively.