The requirements for binding at the N-acetyl-L-glutamate binding site of carbamoyl phosphate synthetase I were studied by the displacement of the activator from the central enzyme complex by analogs. Two carboxyls are essential and the acetamido group, if linked to the a-carbon, enhances binding 5000-fold. The subsite for the 6-carboxyl is mobile with respect to that for the a-carboxyl. Mixtures of complementary fragment of acetylglutamate do not bind, indicating a strong 'chelate' effect. Substituents revealed the existence of steric constraints around the 6-carboxyl, the a and y-carbons, and the whole of the acetamido group. However, phenyl substituents at the P-carbon did not hamper binding, indicating that substituents at the P-carbon face the solution. This is consistent with binding of acetylglutamate as the minimum-energy conformer. All analogs binding with high affinity are activators. Some analogs that bind poorly are competitive inhibitors. They appear to bind preferentially to a low-affinity conformation adopted by the site when the products dissociate and the substrates bind. The acetamido group plays no role in the binding of the inhibitors but it is crucial for the binding of the activators, and the high-and low-affinity conformations of the site differ markedly in structural selectivity. from ATPA which is the ATP molecule yielding Pi and ATPB is the ATP molecule yielding carbamoyl phosphate) is considerably greater than for the free enzyme [2, 6, 71. The stereospecificity indicates that at least three of the groups attached to the asymmetric a-carbon of acetylglutamate are involved in the binding. Information about the specific role of these groups should be of value in designing agonists and antagonists of acetylglutamate and in mapping the binding site, which is located in the 20-kDa COOH-terminal domain of the enzyme [8].In the present work, binding studies with analogs of acetylglutamate, in which the groups attached to the asymmetric carbon have been systematically modified, have provided acetylglutamate