Aspartate transcarbamylase is a large (310 kD), multisubunit protein that binds substrates cooperatively and undergoes a large change in quaternary structure when substrates bind. The forces that drive this transition are poorly understood. We evaluated the electrostatic component of these forces by using finite difference and multigrid methods to solve the nonlinear Poisson-Boltzmann equation for complexes of the enzyme with several substrates and substrate analogs. The results have been compared with calculations for the unliganded protein. While pK1/2 values of most ionizable residues fall within 3 pH units of values for model compounds, 31 have pK1/2 values that fall outside the range 0-17. Many of these residues are at the active site, where they interact with the highly charged substrate, in the 80s loop or 240s loop or interact with these loops. The pK1/2 values of eight ionizable residues related by the twofold molecular axes differ by more than 3 pH units, providing additional evidence for asymmetry within the crystal. As in the unliganded structure, a set of residues forms a network in which ionizable groups with Wij values greater than 2 kcal-m(-1) are separated by distances greater than 5 A. Some residues participate in this network in both the unliganded and N-phosphonacetyl-L-aspartate (PALA)-liganded structure, while others are found in only one structure. The network is more extensive in the PALA-liganded structure than in the unliganded structure, but consists of two separate networks in the two halves of the molecule.