The role of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways, especially NF-κB-inducing kinase (NIK)-mediated alternative pathway, in CD40-mediated interleukin (IL)-6 and IL-12 productions by immature or mature dendritic cells (DCs) was investigated. Murine myeloid DCs were matured by treatment with lipopolysaccharide. CD40 ligation induced modest or vigorous cytokine productions in immature or mature DCs, respectively. After CD40 ligation, p38 MAPK was significantly activated in either immature or mature DCs. SB203580, a p38 MAPK inhibitor, markedly decreased CD40-mediated IL-6 and IL-12 productions in immature DCs. In mature DCs, SB203580 significantly decreased CD40-mediated IL-6 but not IL-12 production. On the other hand, CD40 ligation induced vigorous activation of the NF-κB alternative pathway including p100 phosphorylation and subsequent nuclear translocations of p52, a processed form of p100, and RelB in mature but not immature DCs. The CD40-mediated phosphorylation of p100 was completely abolished in NIK-mutated mature DCs. The NIK mutation markedly reduced CD40-mediated IL-12 but not IL-6 production by mature DCs. Taken together, we concluded that IL-6 and IL-12 productions in response to CD40 ligation were controlled by p38 MAPK and NIK mediated alternative pathway, respectively, in mature DCs.