A combinatorial tetrapeptide library, Suc-Ala-Phe-Arg-AA₁-OR, in which R=p-formamidobenzyl ester and AA₁ =17 of the 20 natural occuring amino acids, has been synthesized chemically and separated by a reverse phase HPLC. The library was used to study the s-1 subsite specificity of various proteases. The preferred substrate at the s-1 subsite of chymotrypsin is in the order of Trp>Tyr>Phe>Met>Leu. This agreed with the reported data that the favored substrate at the s-1 subsite for chymotrypsin-catalyzed hydrolysis is an aromatic amino acid residue. The hydrophobic amino acid residues at this subsite can be hydrolysized after a longer incubating time. This procedure of selective hydrolysis of a peptide library was used to probe the selectivity of s-1 subsites of four proteases isolated from Bacillus stearothermophilus, subtilisin Carlsberg, subtilisin BPN' and an engineered protease subtilisin 8397. The protease from Bacillus stearothermophilus favored the substrate with residue Lys, and Arg at the s-1 subsite as a trypsin-like protease. The relative reactivities of amino acid residues in the protease-catalyzed hydrolysis of the library can be used as a fingerprint to indentify the protease in a protease family.