Enzymatic acyl-transfer reaction in organic medium competes with the hydrolytic side reaction depending on the water content. The effect of water content on aminolysis activity of alpha-chymotrypsin for the synthesis of Bz-Tyr-Val-NH2 in acetonitrile was examined under the conditions which were devoid of the hydrolytic deacylation. Excess H-Val-NH2 (880 mM) was employed to keep the hydrolysis negligible. The aminolysis rate increased abruptly between 4 and 5% (v/v) water but a further increase in the water content did not affect the reaction rate. This suggests that water added more than 5% (v/v) does not enhance intrinsic enzyme activity but acts only as a nucleophile for the hydrolytic deacylation.