In this study, we investigated whether overexpression of pigment epithelium‐derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT‐PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser‐induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen‐induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization. © 2001 Wiley‐Liss, Inc.