Cathepsin B from brain exhibited both endopeptidase and dipeptidyl carboxypeptidase activity. Recently the factors, contributing to dipeptidyl carboxypeptidase properties of brain cathepsin B, were identified: I. occupation of the enzyme S3 subsite, 2. free C-terminal group of the substrate, 3. specific interaction between the split off dipeptide and the enzyme active site. The identification was carried out using angiotensin I, its C-end tripeptide and chromophore oligopeptides containing p-nitrophenylalanine residue. C-terminal dipeptide was split off in the proopioid peptides dynorphins 1-7 and 1-8, Met-enkephalin-Arg6-Phe7, Met-enkephalin-Arg6-Gly7-Leu8; the enzyme hydrolyzed also the C-terminal dipeptide bond in Leu- and Met-enkephalins without the subsequent hydrolysis of the remaining tripeptide. D-Ala2, D-Leu5-enkephalin were not hydrolyzed; the bond Arg9-Pro10 was resistant to proteolysis in dynorphin 1-11. Cathepsin B split off the C-terminal dipeptide in synthetic substrates Leu-Trp-Met-Arg-Phe-Ala and Trp-Met-Arg-Phe-Ala but not in Met-Arg-Phe-Ala. These results 06.08 M-15 demonstrated the essential role of branched-chain amino acid residue at the position of P2 and/or P3 of substrates for the enzyme dipeptidyl carboxypeptidase activity. The data obtained suggest that Arg residue at the position P2 (dynorphin 1-7) slowed down, D-amino acid at the position P2 (D-Ala2, D-Leu5-enkephalin) and Pro-Lys bond at the position P1-P2 (dynorphin 1-11) inhibited the cathepsin B dipeptidyl carboxypeptidase activity.