In vitro studies have shown hepatocyte growth factor (HGF) to be a potent mitogen for hepatocytes. Direct evidence of a mitogenic role in vivo was sought by inhibiting HGF activity, using continuous administration of neutralizing antibody to rats which had a stimulus for liver regeneration. Alzet osmotic mini-pumps, administering a constant supply of anti-HGF monoclonal antibody (clone D9), were inserted intraperitoneally into male Wistar rats; an irrelevant isotypical antibody was administered to controls. Forty-five animals received an intragastric bolus of 40 per cent carbon tetrachloride (CCl 4 ) and groups of three test and control animals were killed at 24 h intervals for 7 days. Treatment with anti-HGF monoclonal antibody significantly inhibited the levels of immunodetectable HGF in the sera of rats following CCl 4 administration. In comparison with controls, hepatocyte proliferation as assessed by bromodeoxyuridine labelling in anti-HGF-treated animals was significantly inhibited at 24 h (P<0โข001), 48 h (P<0โข001), and 96 h (P<0โข05) post-CCl 4 administration. In contrast, sinusoidal cell proliferation was not significantly different from controls at any time point. Inhibition of the parenchymal proliferative response to acute CCl 4 -induced liver injury by the in vivo neutralization of HGF provides direct evidence that this growth factor plays an important role in liver regeneration following necrosis.