Information on the regulation of urea synthesis in vivo was obtained by examining the relationship between ureagenesis in vivo, citrulline synthesis in vitro, and two factors currently hypothesized to exert short-term regulation of this pathway: the liver mitochondrial content of N-acetylglutamate (NAG) and substrate availability. Rats meal-fed for 4 h every day (4-20 schedule) or for 8 h every other day (8-40 schedule) were used. (1) The citrulline-synthesizing capacity of mitochondria from livers of rats on the 8-40 schedule exceeded the corresponding velocity of urea synthesis in vivo at all time points studied. (2) Mitochondrial NAG in these livers increased from 127 +/- 32 pmol/mg of protein at 0 h to 486 +/- 205 pmol/mg at 3 h after the start of a meal, and decreased thereafter, but the correlation between NAG content and the velocity of citrulline synthesis was not simple, suggesting that NAG is not the only determinant of the state of activation of carbamoyl phosphate synthase I. (3) In rats on the 4-20 schedule killed 1 h after the start of the meal, the liver content of ornithine, citrulline, arginine, glutamate, alanine and urea increased 2.1-12-fold with respect to the values at 0 h; glutamine decreased by 39%. (4) The combined findings indicate that in vivo, moment-to-moment control of the velocity of urea synthesis is exerted by substrate availability. (5) Digestion limits the supply of substrate to the liver, and prevents its ureagenic capacity from being overwhelmed following a protein-containing meal.