In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.