Addition of crown ethers to α-chymotryspin, subtilisin, and otl1er proteases considerably enhances the activity of these enzymes in transesterification reactions of N-acetyl-alanine and -phenylalanine esters in organic solvents. Even much higher enhancements of activity (up to 640 x) are obtained by prior lyophilization of the enzymes in the presence of 18-crown-6. Several possibilities for the origin of the crown ether activation are discussed. Isosteric variation of the leaving ability of the alcoholate group revealed that for more reactive esters the rate of the acylation process becomes determined by a relatively slow physical process, most likely a conformational change of the enzyme and/or displacement of water molecules from the active site. This rate limiting process has important consequences for the enantioselectivity of the enzyme and enables the variation of the enantioselectivity by addition of small amounts of different cosolvents to the organic reaction medium.