The capability of hepatitis B virus (HBV) to increase the transcription of the human hepatic inducible nitric oxide synthase (iNOS) by transactivating its promoter has been studied. We have observed by reverse-transcription polymerase chain reaction (RT-PCR) that although the mRNA for the iNOS was almost undetectable in the human hepatoblastoma cell line, HepG2, it was constitutively expressed in the 2.2.15 cell line (a derivative of the HepG2 that produces complete HBV particles). Transfection of HepG2 and 2.2.15 cells with the p1iNOS-CAT plasmid (containing a 1.1-kb fragment of the iNOS promoter) resulted in an increase in chloramphenicol acetyl transferase (CAT) activity in 2.2.15 cells. Similar results were observed when HepG2 and Chang liver cell lines were cotransfected with the p1iNOS-CAT plasmid and the complete HBV genome. It was shown that pX was responsible for the transactivation by cotransfection of HepG2 cells with the p1iNOS-CAT and plasmids expressing the HBV-encoded pX protein, core antigen, and e antigen. Cotransfection of HepG2 cells with the pX expression plasmids and a series of deletion mutants of the 1.1-kb iNOS promoter fragments established that transactivation by pX depends on the presence of at least one nuclear factor-κB (NF-κB) binding site. This was further confirmed by cotransfecting cells with a plasmid expressing the NF-κB inhibitor, IκB.