Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate from glutamine, bicarbonate, and 2 mol of MgATP. The heterodimeric protein is composed of a small amidotransferase subunit and a larger synthetase subunit. The synthetase subunit contains a large tandem repeat for each of the nucleotides used in the overall synthesis of carbamoyl phosphate. A working model for the three-dimensional fold of the carboxy phosphate domain of CPS was constructed on the basis of amino acid sequence alignments and the X-ray crystal structure coordinates for biotin carboxylase and D-alanine:D-alanine ligase. This model was used to select ten residues within the carboxy phosphate domain of CPS for modification and subsequent characterization of the kinetic constants for the mutant proteins. Residues R82, R129, R169, D207, E215, N283, and Q285 were changed to alanine residues; residues E299 and R303 to glutamine; and residue N301 to aspartate. No significant changes in the catalytic constants were observed upon mutation of either R82 or D207, and thus these residues appear to be nonessential for binding and/or catalytic activity. The Michaelis constant for ATP was most affected by modification of residues R129, R169, Q285, and N301. The binding of bicarbonate was most affected by the mutagenesis of residues E215, E299, N301, and R303. The mutation of residues E215, N283, E299, N301, and R303 resulted in proteins which were unable to synthesize carbamoyl phosphate at a significant rate. All of the mutations, with the exception of the N301D mutant, primarily affected the enzyme by altering the step for the phosphorylation of bicarbonate. However, mutation of N301 to aspartic acid also disrupted the catalytic step involved in the phosphorylation of carbamate. These results are consistent with a role for the N-terminal half of the synthetase subunit of CPS that is primarily directed at the initial phosphorylation of bicarbonate by the first ATP utilized in the overall synthesis of carbamoyl phosphate. The active site structure appears to be very similar to the ones previously determined for D-alanine:D-alanine ligase and biotin carboxylase.