In order to rationalize our discovery of a marked dependence of subtilisin's enantioselectivity on the organic solvent used as the reaction medium, we employed the X-ray crystal structure of the enzyme and the means of interactive computer modeling to construct the structures of the reactive enzyme-substrate complexes. For subtilisin-catalyzed trans-esterifications between vinyl butyrate and S and R enantiomers of chiral secondary alcohols XCH(OH)Y, the computer simulation data clearly explain a higher reactivity of the former enantiomer on the basis of severe steric hindrances experienced by the latter enantiomer in the active site of subtilisin. The models of binding derived by computer modeling also successfully predicted changes in subtilisin enantioselectivity as a function of the sizes of the X and Y substituents in the nucleophile and upon addition of certain inhibitors.