Vascular endothelial growth factor (VEGF) induces the proliferation of endothelial cells and is a potent angiogenic factor that binds to heparin. We have therefore studied the effect of heparin upon the interaction of VEGF with its receptors. Heparin, at concentrations ranging from 0.1 to 10 micrograms/ml, strongly potentiated the binding of 125I-VEGF to its receptors on endothelial cells. Scatchard analysis of 125I-VEGF binding indicates that 1 microgram/ml heparin induces an 8-fold increase in the apparent density of high affinity binding sites for VEGF, but does not significantly affect the dissociation constant of VEGF. Cross-linking experiments showed that heparin strongly potentiates the formation of the 170-, 195- and 225-kDa 125I-VEGF-receptor complexes on endothelial cells. At high 125I-VEGF concentrations (4 ng/ml), heparin preferentially enhanced the formation of the 170- and 195-kDa complexes. Preincubation of the cells with heparin, followed by extensive washes, produced a similar enhancement of subsequent 125I-VEGF binding. The binding of 125I-VEGF was completely inhibited following digestion of endothelial cells with heparinase and could be restored by the addition of exogenous heparin to the digested cells. The enhancing effect of heparin facilitated the detection of VEGF receptors on cell types that were not known previously to express such receptors. Our results suggest that cell surface-associated heparin-like molecules are required for the interaction of VEGF with its cell surface receptors.