A possible method for the determination of amino acid sequence of polypetides involves their degradation into dipeptide fragements with dipeptidyl amino peptidase I (Cathepsin C). The action of the enzyme was characterized on different model paptides of known structure. Some tryptic paptides of rabbit muscle aldolase were isolated by gel filtration on Sephadex G-25 column and purified by electrophoretic and chromatographic methods. These peptides and synthetic peptide ACTH1-32 were subjected to dipeptidyl aminopeptidase I digestion. The yield of the dipeptides were reasonable good and the structure of the dipeptides was determined by dansyl Edman method, thin layer ion exchange chromatography and high voltage electrophoresis. The action of dipeptidyl aminopeptidase I (Cathepsin C) was completely inhibited by internal prolyl residues.