Ischemic eye disease often results in ocular neovascularization, presumably due to the elaboration of growth factors. Diabetic retinopathy is a classic example in which dramatic retinal neovascularization arises after ischemic retinal damage. The characterization of vascular endothelial growth factor (VEGF) as an angiogenic molecule whose expression is markedly induced by hypoxia makes it a promising candidate for mediating ischemic retinal neovascularization. Thus, we have characterized the structure, binding, and regulation of VEGF receptors in bovine retinal (BREC) and aortic endothelial cells (BAEC). VEGF stimulated a 2.1-fold increase in BREC number and DNA content at 0.6 nmol/l VEGF (P < 1 × 10−7). Scatchard binding analysis demonstrated specific high-affinity VEGF receptors on BREC with a Kd of 4.9 ± 0.6 × 10−11 mmol/l, similar to that observed for BAEC at 5.1 ± 0.4 × 10−11 mmol/l. BREC, however, possess 1.5 × 105 high-affinity receptors/cell, threefold more than BAEC (P < 0.003) and more than any cell type reported previously. 125I-VEGF affinity cross-linking revealed complexes at 220 and 170 kDa in BREC, but only a 220-kDa band of lesser intensity in BAEC. Cross-linking was displaceable in a dose-dependent manner by VEGF (P < 0.01) but not by other hormones. Hypoxia increased VEGF receptor number 50% in BREC without altering affinity. Antiphosphotyrosine immunoblotting showed VEGF-stimulated tyrosine autophosphorylation of VEGF receptor bands at 225 and 220 kDa and another band at 80 kDa within 1 min. These findings suggest that VEGF may mediate retinal vascular proliferation through large numbers of high-affinity receptors on retinal vascular endothelial cells and suggest that VEGF may be an important mediator of neovascularization induced by ischemic retinopathies such as diabetes.