Binding between immunoglobulin light chain (LC) and Tamm-Horsfall glycoprotein (THP) triggers cast formation in myeloma nephropathy. The binding site on THP has been localized to peptide fragment AHWSGHCCL (225-233) (J. Invest. Med, 43:263A, 1995). The present study performed a structural analysis of peptide 225-233. Binding interaction of each modified peptide fragment with LCs was examined by observing the ability of these fragments to compete with human THP for binding to LCs. Three nephrotoxic LCs were used in this study. Peptide AHWSG did not interact with LCs, although removal of AHWS from peptide 225-233 decreased binding affinity. Replacement of cysteine(s) with serine(s) decreased binding ability dramatically. HPLC analysis of peptide 225-233 demonstrated multiple peaks, indicating different structures of this peptide fragment caused by the two cysteine residues. Purification of the isoforms of this peptide and characterization using mass spectroscopy and Ellman's reagent showed that only the isoform possessing intramolecular disulfide bond interacted with the LCs. Chemical modification of the sulfhydryl groups also abolished interaction with LCs. There was no obvious change in binding ability with LCs after histidine 230 was replaced by serine. Peptide 225-233 synthesized using D-amino acids also interacted with LCs. Conclusions: 1) Peptide GHCCL is critical for binding with LCs, while addition of the other amino acid residues, AHWS, facilitates binding; 2) intramolecular disulfide bonding between cysteine 231 and 232 is necessary for binding with LCs; 3) peptide 225-233 composed of D-amino acids may possess clinical efficacy in preventing cast nephropathy.