Abstract Background/Aims A major role has been described for inducible nitric oxide (NO) synthase in several chronic inflammatory liver diseases. N-Acetyl-cysteine (NAC) is a sulfhydryl donor molecule with antioxidant and antiinflammatory effects. It attenuates NO generation following lipopolysaccharide injection in rats. Our goal was to study the effect of NAC on NO synthase induction in hepatocytes in response to proinflammatory cytokines. Methods The effect of NAC on NO synthase induction was studied in the human hepatocyte cell lines HepG2 and 2.2.15 treated with a mixture of proinflammatory cytokines. Interactions between NAC and cytokines on nuclear factor-κB (NF-κB) activation and NO synthase promoter transactivation were investigated. Results NAC dose-dependently modulated the induction of NO synthase mRNA expression, the release of nitrites and the formation of NF-κB binding complexes in cytokine-treated hepatocytes. NAC also reduced the transactivation of the NO synthase promoter. Conclusions Our data show that exposure of hepatocytes to NAC modulated NO synthase expression and NF-κB activity, the key responses of the hepatocyte to inflammatory mediators. These data constitute preliminary evidence that NAC might have hepatoprotective actions of potential relevance in chronic inflammatory liver diseases, mediated partially through the modulation of NO production.