Based on the knowledge that adenovirus (Ad)-mediated expression of the murine gp75 melanoma antigen (Adgp75) will effectively immunize mice against H2-matched B16 melanoma cells, probably via cell- mediated immune mechanisms, we hypothesized that Ad-mediated delivery of the murine interleukin-12 (IL-12) complementary DNA heterodimer would have independent therapeutic effects on tumor growth, and that the combination of the two vectors would work synergistically to augment the antitumor response. We evaluated the therapeutic effect of each vector alone and in combination for efficacy in C57BL/6 mice with preestablished (2 d) B16 melanoma-derived pulmonary metastases, using the number of lung metastases as the efficacy parameter. Intraperitoneal administration of Adgp75 (10(8) PFU) reduced tumor burden to 45 +/- 7% of controls (P < 0.01), and AdIL12 administration (10(8) PFU, intraperitoneally) reduced the number of metastases to 43 +/- 7% of controls (P < 0.01). The combination of Adgp75 (10(8) PFU, intraperitoneally) and AdIL12 (10(8) PFU, intraperitoneally) provided further protection (15 +/- 3%; P < 0.01 as compared with naive control; P < 0.01 compared with Adgp75 or AdIL12 alone). Mice receiving AdIL12 showed increased natural killer cell (NK cell) function in an in vitro cytotoxicity assay, with a dose- dependent lysis of YAC-1 cells and, to a lesser extent, lysis of B16 cells. To assess the relative contribution of major histocompatibility complex I (MHC I) Dependent and Independent activity in combination therapy with Adgp75 plus AdIL12, we performed adoptive transfer experiments, using splenocytes from mice receiving Adgp75, AdIL12, or Adgp75 + AdIL12, from among which NK cells had been selectively depleted in vitro prior to adoptive transfer. Each group showed significant decreases in tumor burden resembling those with primary treatment. Interestingly, NK-cell depletion from among cells derived from the Adgp75- and AdIL12-treated mice significantly altered the therapeutic response (P < 0.01 compared with the Adgp75 + AdIL12 group), suggesting a significant role of NK-cell-mediated cytolysis in vivo, although there was still a significantly reduced tumor burden (P < 0.01 compared with that of naive controls). Collectively, these data support the concept that the combination of AdIL12 and Adgp75 provides additive effects against pulmonary metastases of B16 melanoma by MHC-independent (NK cell) means as well as MHC-dependent cytotoxic lymphocyte means, suggesting that this therapy may be a useful adjuvant in the treatment of metastatic melanoma.