Hepatocyte growth factor (HGF), first identified as a potent mitogen for mature hepatocytes, has been reported to have various activities. We investigated protective effect of continuous HGF supply on carbon tetrachloride (CCl4)-induced acute liver injury in rats. We transfected immortalized but not tumorigenic rat fibroblasts (Rat-1) with an expression plasmid containing the human HGF cDNA and established several cell lines expressing HGF. The biological activity of HGF produced by these cell lines was confirmed by its mitogenic effect on rat hepatocytes in vitro. Either one of the high-HGF-producer cell lines or parental Rat-1 cell line was transplanted into a syngenic rat spleen. Twelve days after transplantation, each rat was intraperitoneally injected with CCl4 and sacrificed 48 h after CCl4 injection. In rats with continuous HGF supply significantly lower serum glutamic-pyruvic transaminase (GPT) level was observed compared to its marked elevation in control rats and the degree of hepatocyte damage was slight on histological analysis. These results indicate that continuous HGF supply effectively inhibits CCl4-induced acute liver injury and may suggest the possibility that this system would be useful on various liver diseases.