Human hepatocytes infected by hepatitis B virus (HBV) produce the proinflammatory cytokine, tumor necrosis factor α (TNF-α). In this study, we explored the mechanism of induction of TNF-α synthesis by HBV. We found that the stable HBV-transfected hepatoma cell line, 2.2.15, expressed high-molecular-weight (HMW) TNF-α mRNAs, which were absent in the parent HepG2 cells. Treatment of 2.2.15 cells with interferon alfa (IFN-α) and/or interleukin-1β (IL-1β) reduced both viral gene transcription and TNF-α mRNA expression. Transient or stable transfection of hepatocyte-derived cell lines with HBV X protein (HBx) expression vectors induced the production of biologically active TNF-α. In these cells, the HBx-induced TNF-α was detected both as cell-associated and soluble forms. Luciferase gene-expression assays showed that the TNF-α gene promoter contained target sequences for HBx trans-activation within the proximal region of the promoter. These results indicate that the hepatocyte TNF-α synthesis induced by HBV is transcriptionally up-regulated by HBx. Thus, HBx may have a role in the induction of the intrahepatic inflammatory processes that take place during acute and chronic hepatitis B.