Insulin-like growth factor binding protein-3 (IGFBP-3) binds to specific membrane proteins located on human breast cancer cells, which may be responsible for mediating the IGF-independent growth inhibitory effects of IGFBP-3. In this study, we evaluated IGFBP-3 binding sites on breast cancer cell membranes by competitive binding studies with IGFBP-1 through -6 and various forms of IGFBP-3, including synthetic IGFBP-3 fragments. Scatchard analysis revealed the existence of high-affinity sites for IGFBP-3 in estrogen receptor-negative Hs578T human breast cancer cells (dissociation constant (Kd) = 8.19 +/- 0.97 x 10(-9) M and 4.92 +/- 1.51 x 10(5) binding sites/cell) and 30-fold fewer receptors in estrogen receptor-positive MCF-7 cells (Kd = 8.49 +/- 0.78 x 10(-9) M and 1.72 +/- 0.31 x 10(4) binding sites/cell), using a one-site model. These data demonstrate binding characteristics of typical receptor-ligand interactions, strongly suggesting an IGFBP-3:IGFBP-3 receptor interaction. Among IGFBPs, only IGFBP-5 showed weak competition, indicating that IGFBP-3 binding to breast cancer cell surfaces is specific and cannot be attributed to nonspecific interaction with glycosaminoglycans. This was confirmed by showing that synthetic IGFBP-3 peptides containing IGFBP-3 glycosaminoglycan-binding domains competed only weakly for IGFBP-3 binding to the cell surface. Rat IGFBP-3 was 20-fold less potent in its ability to compete with human IGFBP-3(Echerichia coli), as well as 10- to 20-fold less potent for cell growth inhibition than human IGFBP-3, suggesting the existence of species specificity in the interaction between IGFBP-3 and the IGFBP-3 receptor. When various IGFBP-3 fragments were evaluated for affinity for the IGFBP-3 receptor, only those fragments that contain the midregion of the IGFBP-3 molecule were able to inhibit 125I-IGFBP-3(Escherichia coli) binding, indicating that the midregion of the IGFBP-3 molecule is responsible for binding to its receptor. These observations demonstrate that specific, high-affinity IGFBP-3 receptors are located on breast cancer cell membranes. These receptors have properties that support the notion that they may mediate the IGF-independent inhibitory actions of IGFBP-3 in breast cancer cells.