The function of dendritic cells (DCs), antigen-presenting cells that can initiate and regulate cellular and humoral responses, is highly influenced by their level of maturation. Immature DCs may be harmful in anti-tumor immunotherapy, because they can induce immunotolerance rather than immunostimulation. In this study, the authors sought to determine the optimal culture conditions for obtaining fully mature DCs. When DCs were cultured in agonistic anti-CD40 monoclonal antibody-immobilized plates, they showed a higher expression of the maturation marker CD83 than DCs cultured without CD40 ligation or those cultured in medium supplemented with anti-CD40 monoclonal antibody. In addition, when interferon-gamma (IFN-&ggr;) was added to the medium, additive up-regulation of CD83 expression was observed. These DCs treated with both maturation signals showed a higher secretion of interleukin-12. To evaluate the capacity of antigen presentation, specific cytotoxic T lymphocytes were generated using autologous DC pulsed with a human lymphocyte antigen-A24–restricted peptide epitope derived from carcinoembryonic antigen. Interferon-gamma–secreting CD8+ T cells were analyzed by flow cytometry using the cellular affinity matrix technology. Dendritic cells, matured with CD40 ligation and IFN-&ggr;, were more efficient at eliciting an antigen-specific T-cell response in vitro than DCs stimulated with anti-CD40 monoclonal antibody or IFN-&ggr; alone. A cytotoxicity assay using carcinoembryonic antigen-expressing tumor cell lines also showed that DCs matured with both signals were more efficient at inducing cytotoxic T lymphocytes. These results demonstrate that DC culture in an anti-CD40 monoclonal antibody-immobilized plate in medium supplemented with IFN-&ggr; has a positive impact on DC maturation and may be optimal for eliciting an antigen-specific T-cell response without the need for CD4+ T-helper epitopes.