Both high molecular weight kininogen (HK) and low molecular weight kininogens (LK) are potent tight binding inhibitors of platelet calpain (Ki = 2 nM), but the molecular basis for the inhibitory function is not well delineated. The amino acid sequences of the calpain inhibitory domain 2 from human and rat HK were compared for homology with the noninhibitory domains from human and rat domain 3 and from domain 2 of rat T-kininogen, and two areas of nonconserved differences were detected. Computer three-dimensional models were constructed on a template built using the x-ray crystallographic data for cystatin, an evolutionary precursor of HK. Two nonconserved regions in the calpain inhibitory domains flank the highly conserved motif QVVAG to form a continuous surface for interaction with cysteine proteases. Three peptide sequences, components of the modeled surface, were chosen for synthesis from HK D-2: VHPISTQSPDLE (peptide 146-156, NH2-terminal), CTDNAYIDIQLRIASFSQNC (peptide 229-248, COOH-terminal), and CQRQVVAGLNFRIC (185-189, central) containing QVVAG. This last peptide differs from the natural sequence by substitutions of A185C and T195C. Peptides 185-198 and 229-248 were folded by air oxidation of their cysteine residues and then tested for their ability to inhibit calpain and papain. The folded peptide 229-248 inhibited calpain with an IC50 35 microM and unfolding reduced this effect. The folded peptide 185-198 did not inhibit calpain, but when preincubated with calpain, could block the inhibition by HK indicating a probable enzyme binding site. Peptide 146-157 did not inhibit calpain but could inhibit papain with an IC50 of 20 microM. We have thus defined separate binding and inhibitory sequences on HK which form a contiguous surface for thiol protease interactions.