Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-α signal through both the IκB/NF-κB and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF-α-induced NF-κB and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF-α-induced, but not IL-1β-induced, IκBα degradation and NF-κB activation in NIH-3T3 and IEC-6 cells. TNF-α-induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-α was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1β-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF-α-mediated signaling through IκB/NF-κB in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1β signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.