Objective: To observe the therapeutic efficiency of adenovirus-mediated interleukin-12 gene(AdmIL-12) and CD40 ligand gene(AdmCD40L) intratumoral transfer in established murine melanoma in vivo. Methods: C57BL/6 mice were inoculated subcutaneously with B16 cells to establish the murine melanoma model. The tumor-bearing mice were injected intratumorally with murine IL-12 gene and CD40L gene recombinant adenovirus. Tumor growth and the survival of tumor-bearing mice were observed. The CTL activity was measured in vitro by lactate dehydrogenase(LDH) release assay. Results: Both AdmIL-12 and AdmCD40L can be efficiently expressed in vitro and in vivo. The treatment with AdmIL-12 could significantly inhibit the tumor growth and prolong the survival period of the tumor-beraing mice. Splenic CTL activity of the mice was also enhanced after IL-12 gene transfer. But the anti-tumor effects of AdmCD40L gene were not significant. In contrast, Co-delivery of IL-12 gene and CD40L gene lead to stronger antitumor effects than IL-12 gene alone. Conclusion: Adenovirus-mediated interleukin-12 gene and CD40 ligand gene transfer together intratumorally has significant therapeutic effects on mice melanoma in vivo.