Ten peptides were tested as substrates for cathepsin I. The enzyme exerted both endopeptidase and aminopeptidase activities on 5 substrates, only aminopeptidase activity on 3 others, and only endopeptidase cleavage on one compound. One peptide was not significantly hydrolyzed. Aminopeptidase activity stopped one residue before a proline residue and endopeptidase cleavage took place one residue after a proline residue. With these substrates, this enzyme appears to have a broad specificity in its aminopeptidase action. However, cathepsin I appears to have a much narrower and more specific endopeptidase activity, hydrolyzing peptide bonds involving the nitrogen of branched chain or bulky or hydrophobic amino acids. Finally, some differences in the physical, chemical and biological properties of cathepsins H and I were discussed.