The progress of enzymatic peptide synthesis catalyzed by alpha-chymotrypsin and subtilsin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH(2) (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the L-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH(2) or ZAlaAlaPheXaaNH(2) occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S’(1)-specificity of alpha-chymotrypsin and the S-1-specificity of subtilisin 72, while the S-1-specificity of alpha-chymotrypsin and the S’(1)-specificity of subtilisin 72 were not affected . When the DMF content was increased, the rate of the alpha-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions Hydrolysis of the acyl-group donor did not occur in the alpha-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation ofMalAlaAlaPheOH was observed at the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.