The binding of the 165 amino-acid form of vascular endothelial growth factor (VEGF165) to the VEGF receptors of vascular endothelial cells was potentiated by heparin and heparan-sulfate, but not by other glycosaminoglycans. Heparin fragments of 16-18 sugar units inhibited the binding of 125I-VEGF165 to VEGF receptors, while fragments larger than 22 sugar units potentiated the binding. Over-sulfated heparin was a better potentiator of 125I-VEGF165 binding than native heparin. O-desulfated and N-desulfated heparins potentiated the binding to a lesser extent than native heparin. Heparin and N-desulfated heparin efficiently inhibited the binding of 125I-VEGF165 to alpha 2-macroglobulin, but surprisingly, O-desulfated heparin was an ineffective inhibitor. Since alpha 2-macroglobulin does not bind heparin, it follows that VEGF165 does not bind O-desulfated heparin efficiently. These results suggest that the mechanism by which heparin modulates the binding of VEGF165 to the VEGF receptors may require an interaction with cell surface heparin binding molecules.