The present paper reports on studies concerned with furnishing evidence for peptide synthesis in the course of in vitro proteolysis. To this end, the oxidized chain B from insulin (INS) (S = 5 % in demineralized water) was subjected to tryptic proteolysis (E/S= 1/50; pH 5; 37°C;24 h). HPLC - as well as amino acid - and sequence analytical studies have shown that the heptapeptide INS 23-29 (Gly-Phe-Phe-Tyr-Thr-Pro-Lys) liberated by way of hydrolysis is linked by tryptic synthesis via transpeptidation or condensation to form a dimer which accounts for 15% of the amount of monomer. The results of the model trials show clearly that during in vitro proteolysis chemical reactions beyond hydrolytic cleavage of peptide bonds take place. In principle, plastein-like reactions (transpeptidation, condensation) can occur during each in vitro proteolysis.