alpha-Chymotrypsin (CT) was lyophilized from an aqueous solution in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CyD). The enzyme preparation was used as a catalyst for transesterification between N-acetyl-L-tyrosine ethyl ester and methanol in a mixed solvent of acetonitrile/water (97/3 (v/v)). The enzyme preparation had much higher catalytic activity than free CT. The activity increased with an increase of HP-beta-CyD/CT ratio and reached a maximum activity at the weight ratio of 4. Also, the activity of HP-beta-CyD/CT increased with an increase in water content in the reaction media, and the maximum activity was obtained at 5-10% water. The fluorescence spectroscopic analysis suggested that the co-lyophilization with HP-beta-CyD increased the structural stability of CT in acetonitrile/water. Upon co-lyophilization with HP-beta-CyD, the activity of CT increased in any of the solvents used, but the activity depended strongly on the nature of the organic solvents. The catalytic activity of subtilisin Carlsberg (STC) also increased by co-lyophilization with alpha-, beta-, -gamma-CyD or tri-O-methyl-beta-CyD. alpha-CyD gave the best result, while HP-beta-CyD diminished the activity of STC.