A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a "one bead, one peptide" combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P2, P1 and P'2) with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated alpha-chymotrypsin under appropriate conditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with Ki values in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed.