We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.