Purpose: Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a valuable tool for alternative treatment of ocular diseases. Cellular specificity of transgene expression could be influenced by either the viral capsid or the choice of promoter. The use of cellular promoter, cathepsin D (CatD) proximal promoter, and its potential for application in rAAV-based gene therapy are evaluated in this study. Materials and methods: Different sizes of CatD proximal promoter fragments -769 to -1 (CD768), -366 to -1 (CD365), -253 to -1 (CD252), and -124 to -1 (CD123) were subcloned upstream of the green fluorescent protein (GFP) gene. The specificity and activity of the promoter were tested in vitro using human retinal pigment epithelium (RPE) cell lines (RPE51, D407), with the human fibroblast cell line (F2000) used as control. The promoter fragment that showed higher activity in RPE cells was chosen to generate rAAV vector based on AAV scrotype 2. The ability of CatD promoter to target transgene expression to RPE in vivo was determined following subretinal delivery of rAAV particles into nonpigmented RCS/rdy(+) rats. Results: In vitro studies showed that the proximal promoter fragment CD365 targeted high GFP expression in RPE cells. This fragment was then used to generate the AAV.CD365.gfp construct. It was shown in vivo that following subretinal injection, the CD365 fragment in AAV.CD365.gfp directed GFP expression preferentially into RPE cells. Relatively lower level of GFP expression was detected in the neuroretina. In contrast, injection of control virus (AAV.CMV.gfp) resulted in equal levels of transduction and fluorescence signal intensity in both the RPE and photoreceptor cells. Conclusions: The results of our study demonstrate that the promoter fragment CD365 has the potential to target preferential gene expression in the RPE following subretinal injection in rAAV-mediated gene therapy.