The 165 amino acid form of vascular endothelial growth factor (VEGF₁₆₅) is a heparin-binding growth factor with mitogenic activity for vascular endothelial cells. We examined activities of various heparin derivatives toward their interactions with VEGF₁₆₅ using an enzyme-linked immunosorbent assay and elucidated the structural features in heparin for the interactions. Native heparin interacted with VEGF₁₆₅, whereas N-desulfated, N-acetylated (N-DS, N-Ac-) heparin, and 6-O-desulfated (6-O-DS-) heparin did not. The 2-O-desulfated (2-O-DS-) heparin retained the ability for the interaction with VEGF₁₆₅. In contrast, the 2-O-DS-heparin exhibited no ability for the interaction with FGF-2 and HGF. Thus, structural requirements in heparin for the specific interaction with VEGF₁₆₅ are distinct from those with FGF-2 and HGF which require a high content of 2-O-sulfate groups. In a cell proliferation assay, native heparin and 2-O-DS-heparin exhibited inhibitory abilities for VEGF₁₆₅-induced proliferation of human umbilical vein endothelial cells (HUVECs) with their high concentrations (more than 64 µg/ml), while only native heparin could enhance the proliferation of the chlorate-treated cells. These results suggested that a high content of 2-O-sulfate groups is not required for the specific interaction with VEGF₁₆₅ alone, although it is essential for the mitogenic activity of the growth factor.