A cytotoxicity model of primary cultured hepatocytes with D-galactosamine (D-Gal) has been established. The ALT value of culture medium was used as the marker of cell injury. Using this model, the in vitro antihepatotoxic effects of hepatic stimulator substance (HSS), isolated from regenerating adult rat liver, as well as Potenlin and 15-amino acid solution were observed. The results showed that although HSS could stimulate DNA synthesis in serum-free culture of adult rat hepatocytes, it had no effect to reduce ALT value in vitro. However the latter could be reduced by Potenlin or 15-amino acid solution in high concentrations. The described model may be useful for preliminary screening of antihepatotoxic activity of drugs.