Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine-conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco-conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol-3 kinase pathway (PI3K) and nuclear factor-B were inhibited using LY 294002 and adenoviral overexpression of dominant-negative IB, respectively. The role of p38 and extracellular signalregulated protein kinase mitogen-activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin-D. Apoptosis was determined by measuring caspase-3, -9, and -8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase-8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCAinduced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF-B activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA-induced mitochondria-controlled apoptosis by activating survival pathways. Supplemental material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/supplmat/index.html). (HEPATOLOGY 2004;39:1563-1573.) C holestatic liver diseases are characterized by the accumulation of toxic bile acids, for example, glycochenodeoxycholic acid (GCDCA), causing damage to hepatocytes and cholangiocytes. Ursodeoxy-cholic acid (UDCA) is used as a treatment for patients with chronic cholestatic liver diseases. In primary biliary cirrhosis, doses of 13 to 15 mg/kg daily of UDCA decrease serum liver enzymes, improve liver histological test results, and delay the time to liver transplantation or death for up to 4 years. However, the mechanisms of the beneficial effect of UDCA in these conditions remain unclear. From in vitro and in vivo studies, it is postulated that UDCA protects cholangiocytes against membrane damage induced by hydrophobic bile acids. Furthermore, UDCA stimulates biliary secretion of bile acids and other toxic compounds. In addition, anti-apoptotic effects of UDCA have been described, such as the inhibition of the mitochondrial membrane permeability transition in hepatocytes, 7,8 leading to prevention of mitochondrial release of cytochrome c. 9 A recent study suggested that TUDCA does inhibit apoptosis by preventing the binding of Bax to mitochondria. Hepatocytes are exposed to many proapoptotic compounds. Therefore, antiapoptotic signaling pathways are important to limit programmed cell death. The antiapoptotic action of UDCA in part may be the result of