Cast nephropathy, or myeloma kidney, is a potentially reversible cause of chronic renal failure. In this condition, filtered light chains bind to a common site on Tamm-Horsfall protein (THP), which is produced by cells of the thick ascending limb of the loop of Henle. Subsequent aggregation of these proteins produces casts that obstruct tubule fluid flow and results in renal failure. In the present study, we used the yeast two-hybrid system to determine the site of interaction of light chains with THP. The third complementarity-determining region (CDR3) of both κ and γ light chains interacted with THP. These findings were confirmed in a series of competition studies using a synthetic peptide that corresponded to the CDR3 region and purified THP and light chains. Variations in the CDR3 sequence of the light chain affected binding. Thus, the current studies increase our understanding of the process of cast formation and provide an opportunity to develop strategies that may inhibit this interaction and prevent the clinical manifestations of myeloma kidney.