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The murine VEGF gene is alternatively transcribed to yield the VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈ isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF^164/164 mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF^120/120 mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF^188/188 mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF₁₆₄, was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.