Enzymes are shown to function in nonaqueous media; however, relatively little information is available on the influence of the organic solvent as well as its associated water content on the properties of the enzymatic transition states. A better understanding of these effects will be useful in developing kinetic models that can then be used to predict optimal solvent and substrate choices for enzymatic reactions in organic media. The influence of the reaction media on soybean peroxidase-catalyzed oxidation of para-substituted phenols was studied using Hammett analysis for several organic solvent systems. The catalytic activity and substrate specificity of the enzyme are influenced by the nature of the solvent and its associated hydration. These findings may allow one to draw conclusions about the reaction mechanism and the roles of solvent and solvent hydration on enzyme function.