The interactions between NK cells and dendritic cells (DCs) have been previously demonstrated in vitro. In this report, the in vivo cross-regulation between NK cells and DCs was studied in tumor-bearing mice treated with adenoviral vector expressing IL-12 and agonistic anti-4-1BB Abs. NK cells are essential for both tumor rejection and CTL development in the combination therapy (IL-12 plus anti-4-1BB). The numbers and functional activities of both NK cells and DCs in tumor-infiltrating leukocytes were synergistically increased in the IL-12 plus anti-4-1BB-treated mice compared with treatment with either reagent alone. NK depletion in vivo resulted in a significant decrease in the number of DCs in tumor-infiltrating leukocytes, strongly suggesting that NK cells are involved in the activation and expansion of DCs. The mechanism by which IL-12-activated NK cells regulate DC functions is, in part, mediated through the secretion of IFN-gamma that leads to the up-regulation of 4-1BB by DCs. Furthermore, 4-1BB activation in conjunction with IL-12 gene delivery increased tumor infiltration of green fluorescence protein-labeled DCs and enhanced their MHC class II expression. The activation of DCs by NK cells and the subsequent development of antitumoral CTL responses facilitated by 4-1BB-activated DCs may account for the synergistic effects observed in the combination therapy in comparison to adenoviral vector expressing IL-12 or anti-4-1BB treatment alone.