BACKGROUND It is generally assumed that unwarranted, excessive neovascularization of the retina and iris is a direct response to a hypoxic retinal environment. Prompted by our previous findings that the potent angiogenic factor, vascular endothelial growth factor (VEGF), is hypoxia-inducible, we used in situ hybridization techniques to examine the thesis that VEGF functions as the link between retinal ischemia and a pathologic, intraocular, angiogenic response. METHODS To gain molecular access to human material representing progressive stages of angiogenic eye diseases, in situ hybridization analysis was carried out on sections of whole globes enucleated at the time of ongoing neovascularization. This methodology identified cells that have up-regulated VEGF expression during natural progression of the indicated diseases. A rabbit model was also used to determine whether experimentally induced retinal ischemia leads to up-regulation of VEGF expression. RESULTS Proliferation of vascular elements in proliferative diabetic retinopathy and neovascularization of the retina and/or iris secondary to central retinal vein occlusion, retinal detachment, and intraocular tumors were always accompanied by induction of retinal VEGF expression. Furthermore, in each case, expression of VEGF was induced only in a particular layer of the retina (either the outer nuclear layer, the inner nuclear layer, or the ganglion cell layer), matching the zones affected by impaired perfusion. In a rabbit model simulating retinal vein occlusion, elevated levels of VEGF mRNA were detected within a few days of experimental induction of retinal ischemia, exclusively in the ischemic region. CONCLUSIONS VEGF may be one of the long anticipated factors linking retinal ischemia and intraocular angiogenesis. Irrespective of the cause of retinal ischemia, sustained overproduction of VEGF by ischemic retinal cells may promote retinal and iris neovascularization in a number of neovascular eye diseases.