The characterization of the S’subsite specificity of native and ethylated alpha-chymotrypsin has been studied via acyl transfer reaction in acetonitrile containing 10 vol% of water. Using Ac-Tyr-OEt as acyl donor, we investigated the partitioning of acyl-chymotrypsins between water and amino acid and peptide-derived nucleophiles. For the investigation of S’(2) subsite specificity, a series of 19 dipeptides of the general structure Ala-Xaa (Xaa represents all natural amino acids except cysteine) were used. From the values of the apparent partition constants P-app, the order of preference for the P’(2) position is estimated to be : positively charged > hydrophilic greater than or equal to hydrophobic > aromatic > Pro > negatively charged side chain. Concerning the S’(1) specificity, the same preference is deduced based on the study with the series of amino acid amides and Xaa-Ala dipeptides. In contrast to the nucleophilic specificity of alpha-chymotrypsin in aqueous solutions, free dipeptides and hydrophilic amino acid derivatives as nucleophiles exhibit much higher reactivities for acyl transfer in acetonitrile. We have not observed a significant difference in nucleophilic specificity between native and ethylated chymotrypsin.