Children presenting with large retinoblastomas are currently treated by enucleation. As most patients are young children, the long-term repercussions of such surgery are often devastating. Subsequent radiation or chemotherapy, although effective in managing residual tumor, greatly increase the probability of the development of second malignancies later in life. Smaller tumors can sometimes be managed with local cryo- or laser surgery, thus saving the eye. The hypothesis that gene therapy could be used to reduce the tumor size sufficiently to allow local control was tested using a murine model of retinoblastoma. Y79Rb human retinoblastoma cells can be killed in vitro when transduced with an adenoviral vector containing the herpes simplex thymidine kinase gene (AdV-TK) followed by treatment with the prodrug ganciclovir. Intravitreal injections of Y79Rb cells in immunodeficient mice produce an aggressive, metastatic murine model of retinoblastoma. When these murine retinoblastomas were transduced in vivo with AdV-TK and the animals treated with intraocular injections of ganciclovir, 70% showed a complete ablation of detectable tumor. Treated animals had a significant prolongation of progression-free survival as compared with untreated controls. Gene therapy effectively reduced the tumor burden in this murine model of retinoblastoma. Thus gene therapy, in conjunction with local surgical control, may provide an effective alternative to enucleation, systemic chemotherapy, or radiotherapy for treatment of large, nonmetastatic retinoblastomas in children.