Abstract This study is the first to demonstrate activation of NF-κB binding just 10 minutes into the commonly employed hepatocyte isolation procedure. It is further reported that the anti-oxidant Trolox can prevent the induction of NF-κB during the well established hepatocyte isolation procedure but not during their subsequent culture. However both phases of NF-κB activation are inhibited by L-NAMEintimating a role for NO production, via nitric oxide synthase. These findings demonstrate that at least 2 different signal transduction pathways are operative during hepatocyte isolation and culture. Thus further studies employing Trolox and L-NAMEwill help delineate how each pathway contributes to the generalised loss of liver function commonly observed in vitro.