We have established a syngeneic mouse tumor model to test the efficacy of the drug-sensitizing enzyme thymidine kinase from herpes simplex virus (HSVtk) in vivo. Activated mutant Ki-ras (G12V) is frequently found in human colon cancer and adenocarcinomas of the lung and pancreas. We have transformed BALB/c-3T3 cells by stable transfection of a plasmid directing the expression of the mutant Ki-ras cDNA. To transfer the HSVtk gene into tumor cells we used a Moloney murine leukemia virus (MoMLV)-based retroviral vector that carries the HSVtk gene. In this study we show that the activity of HSV-TK inhibits tumor growth in immune-compromised nude mice following GCV treatment for up to 50 days but is not sufficient to completely eliminate all tumor cells in these mice as evidenced by the occurrence of tumors between 40 and 50 days after tumor cell implantation. By contrast, immune-competent BALB/c mice develop a long-lasting antitumor immunity in response to HSVtk transduction and GCV treatment, indicating that the immune system is important for the long-term tumor suppression in vivo. In the presence of GCV co-culturing of tumor cells with HSVtk transfected cells leads to the efficient killing of HSVtk negative tumor cells. While this retroviral vector independent HSV-TK/GCV-mediated bystander effect is not sufficient to inhibit tumor formation in athymic animals it is very efficient in immune-competent syngeneic mice. Taken together the data indicate that the antitumor activity of HSV-TK is enhanced by an intact immune system.