['Immunotherapy is often effective only for small tumor burdens and, in many cases, is restricted to subcutaneous tumors. Here, we investigated the antitumor effects of combination therapy with GM-CSF and IL-12 on orthotopic liver tumors with intermediate or large tumor volumes, or on chemically-induced multifocal liver tumors in animals. Adenoviruses encoding GM-CSF or IL-12 were injected intratumorally to animals bearing transplanted tumors, or injected via intrahepatic artery in animals with primary multifocal liver tumors induced by diethylnitrosamine. Our results demonstrated that IL-12, but not GM-CSF, monotherapy displayed significant therapeutic effects, whereas combination therapy with both cytokines displayed synergistic antitumor effects not only on transplanted tumor models with intermediate or large tumor loads, but also on carcinogen-induced multifocal liver tumors. Effector cell analyses, revealed by in vivo cell subset depletion, flow cytometry analysis, and immunohistochemical staining of tumor infiltrates, indicated that NK cells were the prominent antitumor effectors for the IL-12-mediated antitumor activity, whereas CD8+ T cells, NKT cells, and macrophages were more important than NK cells in the combination therapy-mediated antitumor effects. Both IL-12 monotherapy and combination therapy could induce various types of effectors and high levels of IFN-gamma; however, the latter induced much higher levels than the former, which may explain why combination therapy is superior to IL-12 monotherapy.'] Does combined GM-CSF and IL-12 gene therapy synergistically suppress the growth of orthotopic liver tumors?