Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen (Ag)-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-alpha for 7-10 days and stimulated with lipopolysaccharide (LPS). However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class II and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli (IL-1beta,IFN-gamma, TNF-alpha, CpG and LPS) in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-gamma was able to stimulate the maturation of DC2.4 cells. The IFN-gamma-activated ovalbumin (OVA)-pulsed DC2.4 cells have capacity to upregulate MHC class II, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8+ T cell responses and antitumor immunity. Therefore, IFN-gamma-activated immortal DC2.4 cells may prove to be useful in the study of DC biology and antitumor immunity.