The effect of organic solvents on sublilisin Carlsberg catalysis has been investigated with the aid of a thermodynamic analysis. Saturation solubility experiments were performed to provide a quantitative measure of substrate desolvation from the reaction medium. This enabled calculation of the intrinsic enzymic activation energy and resulted in a linear free energy relationship with respect to solvent polarity. The results indicate that the intrinsic activation energy of subtilisin catalysis is lowest: in polar organic solvents, which may be due to transition state stabilization of the enzyme＇s polar transition state for transesterification. (C) 2000 John Wiley & Sons, Inc.