Cytotoxicity of several bile acids to rat cultured hepatocytes was already reported by several authors. However, their reports were investigated under non-physical condition such as albumin free or high concentrations. Furthermore their toxicities were evaluated by different methods. In this study, we investigated the cytotoxicity of various bile acids against cultured rat hepatocytes under the presence of albumin and the physiological concentration. Their cytotoxicities were evaluated by four different parameters. [METHODS] Hepatocytes were isolated from adult male Spraque-Dawley rats and were cultured. One million hepatocytes were plated in 60mm Lux culture dishes with the medium including 5% fetal bovine serum. The medium was changed two hours after plating and hepatocytes were further cultured with 100 μ M of several bile acids such as cholate (CA), chenodeoxycholate (CDCA), deoxycholate (DCA), litocholate (LCA), or taurocholate (TCA) for every 3, 6, 15 or 20 hours at 37 °C. At indicated hours after, the release of lactate dehydrogenase (LDH) from hepatocytes into the medium and Viabilities (Viab) of hepatocytes were assessed after washing three times with culture medium. And hepatocytes in dishes were washed three times with modified serum-free medium (SFM) and hepatocytes were collected with 10% PBS. Their protein concentrations (Pro) were determined, and numbers of hepatocytes (Num) in dishes were also counted.[RESULTS] Cytotoxities of bile acid at 100 μ M increased according to exposure time. The release of LDH into the medium showed good negative correlation between Viab such as r=-0.775. Num showed good positive correlation between Pro such as r=0.661.[CONCLUSIONS] It was suggested that measurement of LDH in the medium is most sensitive to investigate cytotoxicities of various bile acids.