The esterification of N-acetyl-L-tryptophan and N-acetyl-L-tyrosine was studied using α-chymotrypsin as a catalyst in various hydrophilic organic solvents. It was found that both the rate of ester formation and the yield of ester depend largely on the nature of the solvent and water content. The medium effect is discussed in terms of solubility or hydration chymotrypsin. The effects of the buffer solution are also described.