Inhibition of thermolysin-catalyzed peptide synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe-Phe-OMe) in aqueous organic one-phase reaction system by water-miscible alcoholic organic solvents correlated with most of their physicochemical parameters. Structurally similar monoalcohols and diols, including water, showed a linear relationship between inhibition and physicochemical parameters. The structure of organic solvents thus plays the key role in determining enzymatic activity in reaction media containing organic solvents.