Hepatoprotective activity of Phellinus linteus was studied using H(2)O(2)- or galactosamine-injured primary cultures of rat hepatocytes as screening systems. The methanolic extract of the mycelial culture of Phellinus linteus significantly protected against hepatotoxins-induced toxicity in primary cultured rat hepatocytes as seen from the decreased level of glutamic pyruvic transaminase released from the injured hepatocytes. The methanolic extract of the mycelial culture of Phellinus linteus was subsequently fractionated with n-hexane, ethyl acetate, n-butanol and water. Among these fractions, 100 microg/mL of the ethyl acetate fraction was the most active one. The relative protections were 68.9 +/- 5.3% in H(2)O(2)-injured hepatocytes and 46.8 +/- 3.9% in galactosamine-injured hepatocytes, respectively. The ethyl acetate fraction appeared to maintain the glutathione level which was decreased by the treatment of H(2)O(2) or galactosamine and restored the level of RNA synthesis more than two times compared to galactosamine-injured hepatocytes. These results suggest that the ethyl acetate fraction of the mycelial culture of Phellinus linteus protects hepatocytes from H(2)O(2)- or galactosamine-induced injury by maintaining hepatic glutathione level and RNA synthesis as well.