The transition state for subtilisin-catalyzed transesterification was probed by high-pressure kinetic studies in solvents spanning a wide range of dielectric constants. The electrostatic model of Kirkwood described the solvent effects and gave a lower limit of 31 +/- 1.5 Debye for the dipole moment of the transition state, This value remained constant in a wide range of polar and apolar solvents, indicating that the catalytic triad of subtilisin is remarkably robust. Despite the highly polar transition state, substantial rate enhancements relative to the uncatalyzed reaction were measured in highly apolar solvents such as hexane; this is the first report of such an extreme disparity between transition-state and solvent polarities, Moreover, the solvent dependence of the activation volume implies a low effective dielectric of the polypeptide chain in the active site and substantial penetration of the active site by solvent. Kirkwood's model was also used to quantify the effect of an active-site mutation on the transition-state dipole moment. These results illustrate that the electrostatic model combined with high-pressure kinetics can provide unique information on the basic properties of enzyme reaction processes and can be useful in predicting solvent effects on enzyme reaction rates.