The CMV promoter drives high transgene expression and is one of the most commonly used promoters for gene transfer. Tissue-specific mammalian promoters provide an alternative, and it would be useful to have a system to directly compare them to viral promoters free from potential confounding vector-related effects. In this study, we describe how electroporation after subretinal injection of plasmid DNA can be used to perform comparative quantitative analysis of promoter activities. Luciferase assay of eyecup homogenates was carried out after coinjection/electroporation of pGL2, a plasmid containing the promoter fragment of interest coupled to the firefly luciferase gene, and pRL-CMV, a plasmid containing the CMV promoter coupled to the Renilla luciferase gene for normalization. This technique was used to compare activity of different fragments of the 5′-upstream region of the vitelliform macular dystrophy 2 (VMD2) gene, which is selectively expressed in the retinal pigmented epithelial (RPE) cells, and results indicated positive regulatory elements between −104 and −154 bp and between −424 and −585 bp. Addition of a fragment from intron 1 reduced the activity of the −585/+38 bp fragment by 75%. Deletion analysis implicated a 342 bp region near the 5′-end of intron 1 in the repression. Results of transient transfections in two cell lines that constitutively express VMD2 were similar, and results in transgenic mice were consistent, providing validation for promoter analysis by in vivo electroporation. We then explored the time course of expression of the −585/+38 VMD2 promoter fragment and found that compared to cassettes driven by CMV or SV40 promoters, which showed peak luciferase activity on day 2 followed by a rapid decrease in activity, the VMD2 promoter fragment showed lower activity initially, but the activity was sustained for up to 56 days (longest time point measured). A promoter fragment from another RPE-specific gene, Rpe65, showed a similar pattern of sustained expression for at least 112 days. These data indicate that nonviral gene transfer can be used to quantitatively evaluate the activity of promoter fragments independent of influence from viral vectors. A potentially important finding using this new technique is the demonstration that relatively sustained passenger gene expression can be achieved with nonviral gene transfer using mammalian rather than viral promoters.