Selected bile acids activate a nuclear factor-kappa B (NF-kappaB)-dependent survival signaling cascade in cultured hepatocytes. These data suggest that in cholestasis where liver tissue bile acid concentrations are increased, NF-kappaB should be activated and inhibition of NF-kappaB should potentiate liver injury. Our aims were to test these two predictions. Cholestasis was obtained by common bile duct ligation in mice. NF-kappaB activation was demonstrated in nuclear extracts by the electrophoretic mobility gel shift assay from 3-day bile duct-ligated (BDL) mice but not in controls. Immunohistochemistry for NF-kappaB demonstrated nuclear localization in hepatocytes of BDL mice consistent with its activation in this liver cell type. Electrophoretic mobility gel shift assay and immunohistochemistry for NF-kappaB in BDL tumor necrosis factor-receptor 1 knockout mice demonstrated hepatocyte NF-kappaB activation, suggesting that tumor necrosis factor-alpha was not responsible for the activation of this transcription factor. Liver injury was assessed in BDL mice after administration of the adenovirus 5 inhibitor of kappa B superrepressor (Ad5IkappaBsr) to inhibit NF-kappaB. TUNEL-positive cells and serum alanine aminotransferase values were increased at least threefold in mice treated with the Ad5IkappaBsr versus the empty virus. Liver histology also demonstrated increased liver injury in the BDL mice treated with the Ad5IkappaBsr. In conclusion, NF-kappaB is activated in hepatocytes during obstructive cholestasis and functions to reduce liver injury.