In proliferative vitreoretinopathy (PVR), retinal pigment epithelial cells, fibroblasts, or other proliferating cells form contractile membranes in the vitreous cavity of the eye, resulting in traction retinal detachment. Retroviral vector-mediated transfer is a suitable method to transduce the herpes simplex virus thymidine kinase (HSV-tk) gene into proliferating cells in PVR, allowing for the selective killing of these cells. To determine the potential of gene transduction in the environment of the vitreous cavity, we evaluated the effect of vitreous humor on retroviral vector-mediated gene transduction of rabbit dermal fibroblasts in vitro and studied in vivo transduction in rabbit experimental PVR with retroviral vector G1BgSvNa. In addition, we studied the bystander effect in vitro and in vivo in a rabbit model of PVR, with low percentages of HSV-tk-positive cells. Finally, we evaluated the efficacy of intravitreal administration of HSV-tk retroviral vector G1TkSvNa followed by ganciclovir (GCV) in the prevention of experimental PVR. Vitreous humor reduced gene transfer efficiency in vitro in a dose-dependent manner. LacZ expression was found in cells of preretinal or intravitreal membranes of animals of both in vivo and in vitro transduction groups; however, in vivo transduction resulted in a decreased number of transduced cells, with a relative transduction efficiency of approximately 2%. Transduction of HSV-tk was associated with a powerful bystander effect both in vitro and in vivo with significant effects even when HSV-tk-positive cells represented only 1% of the population. In vivo transduction with G1TkSvNa followed by GCV significantly inhibited the development of PVR (p < 0.05). These results suggest that retroviral vector-mediated transfer of HSV-tk into the proliferating cells in PVR may be feasible and may provide a novel therapeutic strategy for this disease.