CD40 signaling modulates the immune response at least in part by activation of nuclear factor B (NFB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFB. Although four members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF3, TRAF5, and TRAF6, bind to the CD40 cyt, how each TRAF protein contributes to the NFB activation by CD40 is not clear. Here we report that TRAF2, TRAF3, and TRAF5 bind cyt-C, whereas TRAF6 binds cyt-N. cyt-N is conserved poorly between human and mouse CD40, while cyt-C is highly conserved. However, single aa substitution of Glu-235 in cyt-N of human CD40 with Ala abolishes the binding of TRAF6 to cyt-N and NFB activation by cyt-N. Conservation of this Glu between mouse and human CD40 strongly suggests that TRAF6 could link cyt-N to signals essential for CD40-mediated immune response. Furthermore, NFB activation by cyt-C is inhibited by a kinase-negative form of NFB-inducing kinase more efficiently than that by cyt-N, consistent with the result that NFB activation by TRAF2 and TRAF5 is inhibited by a kinase-negative form of NFB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFB activating signals emanating from cyt-N and cyt-C are mediated by the different members of the TRAF family and could be regulated in a distinct manner.CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily, which includes TNFR-1, TNFR-2, Fas, lymphotoxin- receptor, CD27, CD30, OX40, and the lowaffinity nerve growth factor receptor (1). CD40 is expressed in late B cells in bone marrow, mature B cells, and certain accessory cells, including bone marrow-derived dendritic cells and follicular dendritic cells (2, 3), and is a receptor for CD40 ligand (CD40L), which is present on activated CD4 ϩ T cells (4). Signaling through CD40 rescues B cells from apoptosis induced by crosslinking of the surface IgM complex (5), induces B cells to undergo Ig isotype switching (6, 7), and activates antigen-presenting cells to prime cytotoxic T lymphocytes (8-10).CD40 signaling events are reported to include modulation of the activity of nonreceptor-type tyrosine kinases such as Lyn, Fyn, and Syk, activation of phosphatidylinositol-3-kinase, phosphorylation of phospholipase C␥2 (11-13), and activation of the Rel͞nuclear factor B (NFB) transcription factors (14). CD40 signaling is also linked to the induction of the B7 (15), intercellular adhesion molecule-1 (16, 17), Fas (18), CD23 (19), lymphocyte function-associated antigen-1 (16), Bcl-X L , Cdk4 and Cdk6 proteins (20). However, the molecular mechanism of signal transduction from CD40 is not clear. The cytoplasmic tail (cyt) of TNFR superfamily members lacks sequences indicative of catalytic activity, but is associated with a family of signal transducer proteins, the TNFR-associated factor (TRAF) family. Among the six members of the TRAF family, TRAF2 (21), TRAF3 (also known as CD40bp, LAP-1, or CRAF1) ...