The major vascular endothelial growth factor (VEGF) isoforms are splice variants from a single gene that differ in their extent of heparin affinity due to the absence of the heparin binding domain in the smallest isoform (mouse VEGF120, human VEGF121). A long-held assumption that has guided the use of VEGF isoforms clinically has been that their differences in heparin binding dictate their ability to diffuse through tissue, with VEGF121 moving most freely and that the distribution of recombinant VEGF would have therapeutically relevant consequences. To test this assumption, we delivered the genes encoding these isoforms by myoblast-mediated gene transfer, a means of delivering genes to highly localized sites within muscle. Surprisingly, all isoforms induced comparable extremely localized physiological effects. Significantly, irrespective of the isoform delivered, the vessels passing within several micrometers of muscle fibers expressing VEGF displayed sharply delineated changes in morphology. The induction of capillary wrapping around VEGF-producing fibers, and of vascular malformations in the muscle at high levels, did not differ among isoforms. These results indicate that heparin binding is not essential for the localization of VEGF in adult tissue and suggest that the preferential delivery of VEGF121 cDNA for clinical applications may not have a physiological basis.--Springer, M. L., Banfi, A., Ye, J., von Degenfeld, G., Kraft, P. E., Saini, S. A., Kapasi, N.K., Blau, H. M. Localization of vascular response to VEGF is not dependent on heparin binding.