IGF-binding protein (IGFBP)-3 is generally considered to have actions that counterbalance those of IGFs and is therefore being developed as a cancer treatment. In breast tumors however, high levels are associated with aggressive tumors and poor prognosis. Consistent with this we have demonstrated that although IGFBP-3 and a non-IGF binding fragment (SPD) reduced attachment and enhanced apoptosis of Hs578T breast cancer cells cultured on collagen or laminin, it promoted their attachment and survival on fibronectin, which is abundant in the matrix of aggressive tumors. We have now examined the factors that determine whether IGFBP-3 has positive or negative actions on breast epithelial cells. IGFBP-3 also promoted survival of Hs578T cells in the presence of an antibody to the 1 integrin subunit or when cholesterol-stabilized complexes were disrupted. These actions were blocked by IGF-I or a MAPK inhibitor. SPD had similar actions on MCF-7 cells that were again reversed on fibronectin, or with disruption of cholesterol stabilized complexes and blocked by the 1 integrin antibody. In contrast IGFBP-3 promoted growth and survival for non-malignant MCF-10A cells, but these effects were again reversed on fibronectin and blocked by the 1-antibody, a MAPK inhibitor or by disruption of cholesterol-stabilized complexes. On Hs578T cells, IGFBP-3 bound to caveolin-1 and 1-integrins, enhancing their aggregation, the recruitment of FAK and the activation of MAPK. In summary, with 3 breast epithelial cell lines, IGFBP-3 had positive or negative effects on growth and survival dependent upon the status of cholesterol-stabilized integrin receptor complexes.