The rational control over enzyme-catalyzed regioselectivity has been studied using sucrose acylation by vinyl esters in organic media as a model. Subtilisins BPN' and Carlsberg preferentially acylate at the 1 '-hydroxyl of sucrose with some acylation observed at the 6-hydroxyl. The preference for the 1'-hydroxyl is strongly affected by the hydrophobicity of the organic solvent and the chain length of the vinyl ester. Increasingly hydrophobic solvents and longer chain lengths lower the favorable formation of the 1'-acylation and improve 6-acylation. Molecular modeling of sucrose in the binding pocket of subtilisin BPN' shows that the 1'-acylation is favored in solvents that can solvate sugars (such as pyridine) as the glucose moiety is exposed to the medium, whereas 6-acylation leaves the entire sucrose molecule buried within the enzyme's binding pocket. Thus, 1 '-acylation is sterically more favorable than 6-acylation. Increasingly hydrophobic solvents affect regioselectivity by changing the degree of solvation of the glucose moiety in the medium and forcing the sucrose 1'-ester completely into the binding pocket. In a related modeling, the vinyl ester chain length was shown to modulate regioselectivity by controlling the bond angles between the resulting acylenzymes and the sucrose thereby affecting the positioning of the sucrose in the binding pocket of subtilisin BPN'. This study shows that control over enzymic regioselectivity can be achieved by rational choices of substrate and solvent.