CD40 is expressed on a variety of tumors; anti-CD40 agonists promote tumor cell apoptosis and subsequent tumor regression. Because the effectiveness of anti-CD40-agonists is dependent upon CD40 surface expression, the current study examined ligation-mediated changes in CD40 protein half-life (t1/2) at the cell surface. This study utilized a CD40+ epithelial cell line (9HTEo-), a CD40 null epithelial cell line (HT-29) engineered to express either wild-type (WT) or mutant (T254A, Q263A, E235A, Δ201) CD40, and the anti-CD40 antibody G28.5. Ligation of endogenous CD40 expressed on 9HTEo- cells decreased CD40 surface protein t1/2 from 13 to 4 h (p <0.05). Ligation of WT-, Q263A-, or T254A-CD40 expressed on engineered HT-29 cells decreased CD40 surface protein t1/2 from an average of 8 to 4 h (p <0.05); T254A and Q263A contain mutated TNF receptor-associated factor (TRAF)2/3-binding sites. In contrast, ligation of E235A and Δ201-CD40 had no affect on its surface protein t1/2 (p <0.05); E235A contains a mutated TRAF6-binding site while Δ201 lacks an intact cytoplasmic tail. These results suggest that anti-CD40 agonists decrease CD40 surface protein t1/2 via a mechanism that involves TRAF6 but not TRAF2/3. The therapeutic implications for CD40-mediated tumor regression are discussed. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.