Action of cathepsin H from bovine spleen on tuftsin, enkephalin, the oxidized B chain of insulin and a variety of synthetic substrates was studied. Cathepsin H splits off only one N-terminal amino acid from each tuftsin and enkephalin, which, according to the literature, led to inactivation of peptides. The enzyme acts on the oxidized B chain of insulin as an aminoendopeptidase: it splits off the N-terminal phenylalanine and the centrally located bond(s). Km and Vmax for the cathepsin H catalyzed hydrolysis of LeuNA, ArgNA LysNA and BANA were determined. Substrates with the free NH2 group were hydrolyzed at a higher rate. Based on the data obtained and the previously reported results on conversion of kallidin into bradykinin, the specificity of cathepsin H and its possible biological functions were discussed. Cathepsin H appears to participate in formation and inactivation of physiologically active peptides. Using the antiserum to spleen cathepsin H it was found that liver, kidney and lung tissues contained the enzymes identical and/or partially identical to cathepsin H from spleen. The data on the properties of cathepsin H from various sources are summarized.