Purpose: Previous studies have demonstrated that adeno-associated virus (AAV) efficiently transduced retinal pigmented epithelial (RPE) cells. The goal of this study was to further evaluate and characterize transgene expression within the RPE cells over time in vivo. Methods: Adeno-associated virus-mediated gene transfer was monitored and quantified by retinal photography following subretinal injection of a recombinant AAV encoding the green fluorescent protein gene (rAAVCMV-gfp) into rat eyes. Retinal function of transduced rat eyes was measured by electroretinography. Results: The maximum level of transgene expression was reached at 8 weeks postinjection followed by a gradual decrease throughout the experimental period. Interestingly, it was observed that while gfp expression was stable in some RPE cells, gfp fluorescent completely disappeared in other cells over the duration of the experiment. The expression of AAV-mediated gfp in RPE cells did not alter the retinal function for over 1 year. Conclusions: These results confirm the importance of this direct visualization system to study vector transgene expression in vivo and support the use of AAV for diseases treatable by targeting RPE cells.