OBJECTIVE Hydrophobic bile acids contribute to hepatocellular injury in cholestasis and rapidly induce apoptosis in vitro; however, unlike Fas agonists, cholestasis does not cause extensive hepatocyte apoptosis. As antioxidants provide protection against bile acid induced liver injury, our premise was that bilirubin, a free radical scavenger with increased plasma levels in the presence of liver disease, could protect hepatocytes against bile acid induced apoptosis. METHODS Freshly isolated rat hepatocytes were incubated for four hours with 100 micromol/l glycochenodeoxycholate (GCDC) alone or with increasing concentrations of unconjugated (UCB) or conjugated (CB) bilirubin. RESULTS Both UCB and CB inhibited GCDC induced apoptosis in a dose dependent fashion and suppressed the generation of reactive oxygen species by hepatocytes. CONCLUSIONS The antiapoptotic effect of bilirubin associated with its antioxidant properties indicates that hyperbilirubinaemia may have a protective role in liver disease.