The diet and nutritional status dominate a tolerance to environmental xenobiotics. In this study, the cytotoxic action of carbon tetrachloride (CCl4) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the dietary carcinogens, was investigated using primary cultured hepatocytes from rats fed a high-fat (23% corn oil) or high-protein (50% casein) diet for three weeks. Both chemicals showed strong cytotoxicity to hepatocytes, which was judged by measurement with the MTT-test and lactate dehydrogenase leakage test. A dietary effect on cytotoxicity was observed; hepatocytes from rats fed the high-protein diet were more susceptible to cytotoxicity than the cells from rats fed a standard diet. On the other hand, ureogenesis, as a cellular function of hepatocytes, was markedly decreased in the cells from rats fed the high-fat diet. These activities were affected in the CCl4-treated cells but not in the Trp-P-1-treated cells. The same trend of both diet and chemical effects was observed in gluconeogenesis from fructose. We conclude that the hepatocytes from rats fed a high-protein diet have high susceptibility to the cytotoxicity of CCl4 and Trp-P-1, but cytotoxicity was not related to the reduction of cellular functions.