The enantioselectivity for subtilisin-catalyzed hydrolysis of ethyl 2-(4-substituted phenoxy)propionates in an aqueous buffer solution was improved by addition of DMSO (54-56% v/v). On the basis of the conformational change of subtilisin Carlsberg observed for FT-IR and CD spectra, the high enantioselectivity for subtilisin-catalyzed hydrolysis of racemic ethyl 2-(4-ethylphenoxy)propionate could be related to a partial decrease of the tertiary structure of the enzyme protein arising from an increase of the ratio of DMSO in the reaction medium. This mechanistic model for the enantiorecognition can also be supported by the discussion based on the value of the Michaelis constant (K-m) obtained for each enantiomer of the substrate.