Among extremely low birth weight survivors, ROP is an important morbidity involving pathologic retinal neovascularization. In animal models, spatial and temporal retinal expression of VEGF demonstrates an important role in both normal vascularization and experimental neovascularization. Purpose: To demonstrate the spatial expression of VEGF mRNA in acute human ROP. Methods: Autopsy eyes from two infants were studied: one with acute ROP; and one who expired with regressed ROP, 4 weeks following cryotherapy. After formaldehyde fixation and paraffin embedding, in situ hybridization was carried out on horizontal sections with an 35S labeled 590bp rabbit cRNA to VEGF. Sense and antisense probed sections were counterstained, photographed at high power, and images aligned for analysis and visual imaging. Findings were contrasted with studies in the kitten model. Results: Spatial variation of VEGF mRNA expression was observed in the ganglion cell layer (GL) where retinal vessels normally grow. Expression was low in vascularized portions of the GL, dramatically upregulated just anterior to the neovascular ridge of ROP tissue in the avascular GL, and absent from extraretinal neovascular membranes. Signal decreased gradually towards the periphery in the GL of the avascular retina (where the retina was still immature) well anterior to the ridge of active ROP. In the post cryotherapy eye all remaining retina was vascularized and GL VEGF mRNA expression was low; no expression over background was seen in the areas of cryosurgical scars or residual neovascular membranes. Baseline mRNA expression was observed in the retinae of both infants throughout the inner nuclear layer and retinal pigment epithelium. Conclusions: Retinal VEGF mRNA expression in human ROP is consistent with the kitten model of this disease, and supports the hypothesis that avascular hypoxic neuroretina upregulates VEGF mRNA expression in human retina, and probably plays an important role in ROP as well as the development of normal retinal vascularization.