Sustained activation of NF-kappaB is one of the causative factors for various liver diseases, including liver inflammation and hepatocellular carcinoma (HCC). It has been known that activating the NF-kappaB signal by hepatitis B virus X protein (HBx) is implicated in the development of HCC. However, despite numerous studies on HBx-induced NF-kappaB activation, the detailed mechanisms still remain unsolved. Recently, p22-FLIP, a cleavage product of c-FLIP.sub.L, has been reported to induce NF-kappaB activation through interaction with the IkappaB kinase (IKK) complex in primary immune cells. Since our previous report on the interaction of HBx with c-FLIP.sub.L, we explored whether p22-FLIP is involved in the modulation of HBx function. First, we identified the expression of endogenous p22-FLIP in liver cells. NF-kappaB reporter assay and electrophoretic mobility shift assay (EMSA) revealed that the expression of p22-FLIP synergistically enhances HBx-induced NF-kappaB activation. Moreover, we found that HBx physically interacts with p22-FLIP and NEMO and potentially forms a ternary complex. Knock-down of c-FLIP leading to the downregulation of p22-FLIP showed that endogenous p22-FLIP is involved in HBx-induced NF-kappaB activation, and the formation of a ternary complex is necessary to activate NF-kappaB signaling. In conclusion, we showed a novel mechanism of HBx-induced NF-kappaB activation in which ternary complex formation is involved among HBx, p22-FLIP and NEMO. Our findings will extend the understanding of HBx-induced NF-kappaB activation and provide a new target for intervention in HBV-associated liver diseases and in the development of HCC.