The expression of VEGF and the relapse-free survival rate of breast cancer patients are inversely related. While VEGF induces the proliferation and migration of vascular endothelial cells, its function in breast cancer cells is not well studied. We reported previously that fibronectin increased VEGF-dependent migration in breast cancer cells. Since VEGF has an extracellular matrix (ECM)-binding domain and possesses binding affinity for heparin, we sought to determine the effects of VEGF in breast cancer cells and the role of heparin and/or fibronectin in VEGF-induced signaling. Cells grown on plastic were compared to those grown on fibronectin or to those grown on plastic in the presence of heparin, and analysed for intracellular signaling, proliferation and migration in response to VEGF(165). Both heparin and fibronectin enhanced the binding of VEGF to T47D cells. After treatment with VEGF, [(3)H]thymidine incorporation, c-fos induction, and the number of migrating cells were significantly higher ( approximately twofold) in cells grown on fibronectin or in cells grown on plastic in the presence of heparin when compared to those grown on plastic only. Likewise, tyrosine phosphorylation of VEGF receptors, MAPK activity and PI3-kinase activity were all several-fold higher in cells seeded on fibronectin or in the presence of heparin as compared to cells exposed to VEGF alone. VEGF-dependent c-fos induction was found to be regulated through a MAPK-dependent, but PI3-kinase-independent pathway. In contrast, the migration of T47D cells in response to VEGF, in the presence of ECM, was regulated through PI3-kinase. Therefore, VEGF requires ECM components to induce a mitogenic response and cell migration in T47D breast cancer cells.