The transport of carbamate through the large subunit of carbamoyl phosphate synthetase (CPS) from Escherichia coli was investigated by molecular dynamics and site-directed mutagenesis. Carbamate, the product of the reaction involving ATP, bicarbonate, and ammonia, must be delivered from the site of formation to the site of utilization by traveling nearly 40 angstrom within the enzyme. Potentials of mean force (PMF) calculations along the entire tunnel for the translocation of carbamate indicate that the tunnel is composed of three continuous water pockets and two narrow connecting parts, near Ala-23 and Gly-575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets were filled with about 21, 9, and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0, and 1.6 kcal/mol, respectively. The release of phosphate into solution at the site for the formation of carbamate allows the side chain of Arg-306 to rotate toward Glu-25, Glu-383, and Glu-604. This rotation is virtually prohibited by a barrier of at least 23 kcal/mol when phosphate remains bound. This conformational change not only opens the entrance of the tunnel but also shields the charge-charge repulsion from the three glutamate residues when carbamate passes through the tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. The mutants retained only 1.7% and 3.8% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS, respectively.