Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture. The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage phiC31 to confer long-term gene expression by means of genomic integration. Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analyzing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was strongest 48 hours after delivery. In the absence of phiC31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4.5-month test period. Eyes injected with phiC31 integrase showed approximately 85-fold higher long-term transgene expression in the retina than eyes without integrase. Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. phiC31 integrase confers robust long-term transgene expression by mediating genomic integration of the transgene. These findings suggest that phiC31 integrase may be a simple and effective tool for nonviral long-term gene transfer in the eye.