We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-κB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-κB in AEC. Results demonstrate that, in AEC, intact NF-κB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-κB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-κB binding and that the activated NF-κB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-κB activation. Findings show that CD40 engagement activated the IκB kinases IKK-α and IKK-β and stimulated IκBα phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-κB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-κB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-κB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.