Dendritic cells (DCs) are professional antigen presenting cells and have key functions in the initiation of immune responses. Hence, antigen-loaded DCs have become important tools for active-specific immunotherapy. In addition to defining strategies for antigen loading, effective T-cell activation by DCs will depend on vaccination protocols that facilitate DC migration to secondary lymphoid tissues and expression of costimulatory molecules and cytokines. Adenoviral gene transfer has been successfully implemented for genetic antigen loading of DCs. In this study, we exploit an adenoviral vector encoding human CD40 ligand (CD40L), Ad5hCD40L, to establish DCs that feature both migration potential and prolonged secretion of the key T-helper 1 cytokine interleukin-12p70 (IL-12p70). Transduction of human monocyte-derived DCs with Ad5hCD40L resulted in efficient CD40L expression, which was detected only intracellularly, and in secretion of IL-12p70. Addition of recombinant interferon (IFN)-gamma shortly after DC transduction substantially increased IL-12p70 secretion. Maturation of DCs was achieved with a standard cytokine maturation cocktail (MC) containing prostaglandin E2 which, however, abolished IL-12p70 secretion by Ad5hCD40L-transduced cells in the absence of IFN-gamma. Only DCs treated with Ad5hCD40L, MC, and IFN-gamma migrated efficiently towards CCL19 and continued to secrete IL-12p70. Finally, DCs transduced with both Ad5hCD40L and an adenoviral vector encoding the melanoma antigen MelanA/MART-1 and treated with MC and IFN-gamma efficiently primed naive autologous CD8+ T cells into antigen-specific cytotoxic T lymphocyte. This strategy to generate DCs that exert both migration capacity and prolonged IL-12p70 secretion after intracellular CD40L expression and IFN-gamma treatment has the potential to further improve current DC vaccination protocols.