Pepsin successfully catalyzed the synthesis of several hydrophobic octa‐ and decapeptides in dimethylformamide‐water solutions containing concentrated urea at pH 4.65. The factors that influence peptide synthesis in the presence of urea aere studied using condensation of the tripeptides Z‐Ala‐Ala‐Phe‐OH and H‐Leu‐Ala‐Ala‐OCH3as a model. The dependence of Z‐Ala‐Ala‐Phe‐Leu‐Ala‐Ala‐OCH3yield on pepsin concentration and pH, as well as the behavior of pepsin during peptide synthesis were studied. It was shown that pepsin catalyzed the synthesis of Z‐Ala‐Ala‐Phe‐Leu‐Ala‐Ala‐OCH3in guanidine hydro‐chloride and sodium dodecyl sulfate solutions. Other proteinases, subtilisin and thermolysin, were applied for the synthesis ofp‐nitroanilides of tri‐ and tetrapeptides in urea solutions.Proteinase‐catalyzed peptide synthesis in the presence of denaturing agents might help to overcome the limitations caused by poor solubility of the starting peptide derivatives, although this effect is sometimes coun