The success of cancer gene therapy is likely to require the targeting of multiple antitumor mechanisms. One strategy involves the use of attenuated, replication-competent virus vectors, such as herpes simplex virus type 1 (HSV-1) mutant G207, which is able to replicate in human tumor cells with resultant cell death and tumor growth inhibition, yet is nonpathogenic in normal tissue. In this study, we demonstrate that infection of established tumors with G207 also induces a highly specific systemic anti-tumor immune response. In a syngeneic, bilateral established subcutaneous tumor model, with mouse CT26 colorectal carcinoma cells in BALB/c mice or M3 melanoma cells in DBA/2 mice, unilateral intratumoral inoculation with G207 caused a significant reduction in the growth of both the inoculated and contralateral noninoculated tumors. This elicited anti-tumor response is dependent on viral infection of the tumor, as intradermal inoculation of G207 in BALB/c mice had no effect on CT26 tumor growth. Treatment of subcutaneous CT26 tumors by intratumoral inoculation of G207 induced a tumor-specific T cell response. CD8+ cytotoxic T lymphocyte (CTL) activity was generated that recognized a dominant "tumor-specific" major histocompatibility complex (MHC) class I-restricted epitope (AH1) from CT26 cells. In immune-competent animals, G207 is acting as an in situ tumor vaccine. Therefore, intratumoral G207 inoculation is able to inhibit tumor growth both by local cytotoxic viral replication in tumor cells and induction of a systemic anti-tumor immune response.