Some properties of carbamoyl-phosphate synthetase (ammonia) were studied in rat-liver mitochondria made selectively permeable by pretreatment with toluene.1 . The Michaelis constants for NH,, MgATP and HCO; were 0.7, 1.2 and 2 mM respectively. N-Acetylglutamate activated the enzyme with a K, of about 0.1 mM. At saturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 inM.2. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase required the presence of both free MgZ + ions and MgATP, and was inhibited by Ca2+ ions and by N-carbamoylglutamate. The known activation of carbamoyl-phosphate synthetase by free Mgz+ is due to an increased affinity of the enzyme for N-acetylglutamate. 3. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase was a slow process : at N-acetylglutamate concentrations below 0.5 mM maxiinal binding was not completed within 30 rnin. The rate of binding increased with increasing N-acetylglutamate concentrations.4. Dissociation of N-acetylglutamate from the enzyme was relatively fast, with a half-time of about 5 min.5. Under all conditions studied there was a close relationship between carbamoyl-phosphate synthetase activity and the amount of N-acetylglutamate bound to the enzyme.6. The data are discussed in relation to the control of carbamoyl-phosphate synthetase in the intact hepatocyte Carbamoyl-phosphate synthetase (ammonia), the first enzyme involved in the biosynthesis of urea in the liver of ureotelic animals, catalyses the formation of 1 mol carbamoyl phosphate from 1 mol ammonia, 1 mol bicarbonate and 2 mol ATP [I]. The enzyme requires both N-acetylglutamate and free Mg2+ for activity [ I , 21 and is inhibited by its products [3] and also by Ca2' [4] and metal ions [5].Carbamoyl-phosphate synthetase is located in the mitochondrial matrix and it represents about 20% of the mitochondrial matrix protein [6-91. Its estimated concentration in the matrix is 0.4 niM [6,7,9] or even higher [8]. This concentration is of the same order of magnitude as the intramitochondrial concentration of its substrate aminonia and of its activators N-acetylglutamate and free Mg2+ (see [lo] for a review).Considering the high intramitochondrial concentration of carbamoyl-phosphate synthetase, one may ask whether kinetic data obtained from studies with the isolated enzyme, used in solution at low concentrations [2,3,8], are applicable to the Ahhreviutions. N-acetylglutamate, N-acetyl-L-glutamate; N-carbamoylglutamate, N-carbamoyl-L-glutamate; CF30PhzC(CN)Z, carbonylcyanide-p-trifluoromethoxyphenylhydrazone.