The effect of multivalent cations on [ 125 I]-IGF binding to cell-associated IGFBPs was investigated using human fibroblasts. The major cell-associated binding site for [ 125 I]-IGF-I is IGFBP-3 and for [ 125 I]-IGF-II are IGFBP-3 and IGFBP-5. Lanthanum and chromium did not affect either [ 125 I]-IGF-I or [ 125 I]-IGF-II binding to cell-associated IGFBPs. By contrast, zinc (Zn 2ฯฉ ), gold (Au 3ฯฉ ), and cadmium (Cd 2ฯฉ ) depressed binding of both ligands. Ligand binding resulted in nonlinear Scatchard plots. Assuming a pre-existent asymmetric model with high-(K aHi ) and low-(K aLo ) affinity sites, Zn 2ฯฉ lowered both K aHi and K aLo . Au 3ฯฉ eliminated K aHi . Assuming that the nonlinear plots were caused by ligand-induced negative cooperativity, Zn 2ฯฉ and Cd 2ฯฉ lowered both K e and K f (affinity of unoccupied and saturated IGFBPs, respectively). Au 3ฯฉ eliminated K e and reduced K f . Zn 2ฯฉ was active at serum levels in lowering IGF binding. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) indicating similar mechanisms of action. A zinc-binding motif is present in the IGFBPs, but not in the IGFs. We demonstrate for the first time that the trace nutrient zinc and related multivalent cations decrease IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF-IGFBP interaction.