Cathepsins S and H were purified from bovine spleen and their catalytic properties compared. The enzymes were shown to be similar by chromatographic properties and by the ability to hydrolyze BzPheValArgNHMec. They could however be distinguished by the fact that cathepsin S reacted with Z-[¹²⁵I]TyrAlaCHN₂ and hydrolyzed Z-PheArgNHMec whereas cathepsin H did not. The substrate and inhibitor specificities of cathepsin H suggest that unlike cathepsins B, L, and S, it cannot accommodate peptides with aromatic side chains in P₂. Cathepsins L and S can accommodate the aromatic side chain of tyrosine in p₁ readily, whereas cathepsins H and B cannot. The specificities of each enzyme for synthetic substrates and inhibitors have enabled the construction of models of the architecture of the active sites of the mammalian cysteine proteinases which clearly show the differences between the four enzymes. A significant characteristic of cathepsin S is that it can hydrolyze insoluble elastin at both acidic and neutral pH; this distinguishes it from all of the other lysosomal proteinases.