The vascular endothelial growth factor (VEGF) family of proteins controls the formation and growth of blood vessels. The most potent and widely expressed isoform, VEGF165, is secreted as a disulfide-linked homodimer with two identical heparin-binding sites. Interactions with heparan sulfate (HS) regulate the diffusion, half-life, and affinity of VEGF165 for its signaling receptors. We have determined a number of key HS structural features that mediate the specific binding of the VEGF165 dimer. Carboxylate groups and 2-O-, 6-O-, and N-sulfation of HS contributed to the strength of the VEGF165 interaction; however, 6-O-sulfates appeared to be particularly important. Cleavage of HS by heparinase, heparitinase, or heparanase severely reduced VEGF165 binding. In contrast, K5 lyase-cleaved HS retained significant VEGF165 affinity, suggesting that binding sites for the growth factor are present within extended stretches of sulfation. Binding studies and molecular modeling demonstrated that an oligosaccharide 6 or 7 residues long was sufficient to fully occupy the heparin-binding site of a VEGF165 monomer. The data presented are consistent with a model whereby the two heparin-binding sites of the VEGF165 dimer interact simultaneously with highly sulfated S-domain regions of the HS chain that can be linked through a stretch of transition sequence.