Successful immunization requires that mature dendritic cells (mDCs) prime T cells in secondary lymphoid tissue (LT). Previously, the authors have shown that LT T cell activation has an increased costimulatory threshold for a proliferative response as compared with peripheral blood (PB) T cells. Therefore, to optimize mDC immunogenicity, DC maturation was studied using LT T cells as responders. While mDCs obtained with soluble CD40Ligand (sCD40L) or a sCD40L/IFN&ggr; combination similarly expressed the CD83 and CCR7 molecules on their membrane, only the latter secreted IL-12. sCD40L/IFN&ggr; mDCs, as compared with sCD40L mDCs, enhanced allogeneic LT T cell proliferation, LT CD4+ cell IFN&ggr; production and LT CD8+ cell cytotoxicity. Enhancement could be predominantly ascribed to IL-12 secreted by sCD40L/IFN&ggr; mDCs and to additional costimulatory signals as shown remarkably in the IFN&ggr; response when IL-12 was neutralized. Therefore, in addition to their membrane phenotype, mDCs to be used in immunization protocols should be assessed for IL-12 secretion as a surrogate marker for an optimum costimulatory potential.