The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.