The biliary excretion of acetaminophen (APAP) is reduced in transport deficient (TR-) hyperbilirubinemic rats lacking the multidrug resistance-associated protein 2 (Mrp2). This mutant strain of Wistar rats has impaired biliary excretion of organic anions and increased hepatic glutathione. The rational for this study was to determine if there is an altered risk for liver damage by APAP in the absence of Mrp2. Therefore, the susceptibility of TR- rats to APAP hepatotoxicity was investigated. Male Wistar and TR- rats were fasted overnight before APAP treatment (1 g/kg). Hepatotoxicity was assessed 24 h later by plasma sorbitol dehydrogenase activity and histopathology. In other studies, TR- rats received buthionine sulfoximine before APAP to reduce hepatic glutathione to values similar to those in Wistar rats. mRNA expression of APAP metabolizing enzymes was also measured in naïve animals. Wistar rats treated with APAP showed significant elevations in plasma sorbitol dehydrogenase activity, while no increases in enzyme activity were observed in TR- rats. Histopathology was in agreement. Hepatic non-protein sulfhydryls were significantly lower in Wistar rats receiving APAP than in TR- rats. TR- rats treated with buthionine sulfoximine and APAP showed dramatic increases in hepatotoxicity. TR- rats had increased mRNA expression of several APAP metabolizing enzymes. Mrp2 expression not only is important in biliary excretion, but also influences the toxic potential of reactive intermediates by controlling intrahepatic GSH and possibly drug metabolism.