The binding sites for high molecular weight kininogen (HK) on prekallikrein (PK) are composed of two discontinuous segments in the primary sequence, one in Apple 1 domain (PK56=F56-G86) and the other in Apple 4 (PK266=K266-G295). The site on HK, HK31, is subsumed in a 31-amino-acid sequence (S565-K595) near the C-terminus which has the same affinity for prekallikrein as the entire HK molecule. The binding among them is likely due to conformational changes which serve to juxtapose the PK binding domain within HK with the HK binding site. Resolution-enhanced Fourier transform infrared spectroscopy (FT-IR) has been employed to analyze the contents of secondary structural elements of PK56 and HK31 and to reveal the possible specific binding portion and structural changes in HK31 and PK56 upon binding. From the amide I bands of their deconvoluted FT-IR spectra, it is known that PK56 contains no helix component, while HK31 has two different helical conformations. A quantitative comparison of the spectra of HK31, PK56 and their binding complex suggests that the conformation of 3(10)-helix in HK31 has been changed to an alpha-helix, and one disordered segment of PK56 may have been changed to extended conformation. The other structural components in PK56 and HK31 remain unchanged. Since previous studies have shown that these peptides mimic the natural protein in their bioactivity, their interaction may reflect similar changes in the natural molecules.