Overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration. In this study, a phosphorothioate oligonucleotide (PS-oligo) targeting both human and rat VEGF(165) genes upstream of the translation initiation code, named DS135 in this study, was evaluated for its uptake dynamics and retinal tolerance after intravitreal (IV) and subretinal (SR) injections in the rhesus monkey. Intravitreal and SR injections of a fluorescent-labeled DS135 (FL-DS135) resulted in both dose- and time-dependent uptake and persistence, and FL-DS135 remained detectable in the retina for at least 3 weeks after injection. Ophthalmic examination showed transient vitreous haze after IV delivery of a high dose but not with a low dose of FL-DS135. Histologic examination showed no evidence of retinal degeneration with respect to IV delivery. After SIR delivery, however, dose-related cellular infiltration, transient residual fluid, and slight distortion of the neuroretina were observed. The biologic efficacy of DS135 was further assessed in a laser-induced CNV model, and development of CNV was determined by fluorescein angiography and histologic examination. Incomplete inhibition of CNV formation was observed after IV and SR injection of DS135, but no statistically significant difference was achieved when compared with dose-matched control of PS-oligo. Analysis of fluorescein angiogram and histologic examination showed less than 30% incidence of CNV development in this monkey model. Our study demonstrated that PS-oligos can be successfully introduced into the retina, although with potential limitations, after SR delivery. DS135, a PS-oligo targeting the VEGF gene upstream of the translation initiation code, partially inhibited CNV formation. An improved CNV model is necessary for further confirmation of the full therapeutic potency of DS135 before clinical application.