A combinatorial library of 400 inhibitors has been synthesized and screened against several serine and cysteine proteases including plasmin, cathepsin B, and papain. The inhibitors are based upon a cyclohexanone nucleus and are designed to probe binding interactions in the S2 and S2' binding sites. This methodology has led to the discovery of inhibitor 15A, which incorporates Trp at both the P2 and P2' positions and has an inhibition constant against plasmin of 5 microM. Data from screening of the library shows that plasmin has a strong specificity for Trp at the S2 subsite and prefers to bind hydrophobic and aromatic amino acids such as Ile, Phe, Trp, and Tyr at the S2' subsite. In contrast, the S2' subsites of cathepsin B and papain do not show a strong preference for any particular amino acid.