The protein BCL-X_L and protein product of proto-oncogene bcl-2 act as apoptosis antagonists, and BCL-X_S serve as a dominant death promoter, including apoptosis following exposure to chemotherapeutic drugs. This investigation examined whether some aspects of the highly integrated process of acetaminophen (AAP)-induced hepatotoxicity involve down-regulation or upregulation of expression of BCL-2, BCL-X_L and BCL-X_S in mouse liver in vivo. Male ICR mice (CD-1; 35–45 g) were treated ip with a hepatotoxic dose of AAP (500 mg/kg) and sacrificed 0, 6, and 18 h later. Blood was collected upon sacrifice for determination of serum alanine aminotransferase (ALT) activity and the liver was sectioned for histopathological diagnosis of necrosis/apoptosis. Portions of liver tissues were also used for DNA extraction (for gel electrophoresis) and Western blot analysis. This study demonstrates that administration of a hepatotoxic dose of AAP to ICR mice results in severe liver injury (ALT leakage >200-fold at 6 h and >600-fold at 18 h) leading to massive cell death by apoptosis (diagnosed by nuclear ultrastructure, histopathology, and DNA ladder), in addition to necrosis coupled with spectacular changes in the BCL-X_L expression (6 and 18 h after AAP administration). Western blot analysis of the liver proteins revealed that mouse liver expresses two proteins, BCL-X_L and BCL-X_S, and does not express BCL-2. As the toxicity progressed, during 6 and 18 h post-AAP administration, the BCL-X_L protein band shifted to a slower mobility band which might represent a phosphorylated form of BCL-X_L. Appearance of this higher molecular weight BCL-X_L protein band correlated with massive apoptotic death of liver cells along with ladder-like DNA fragmentation. In the same time period, death inhibitory gene bcl-2 remained unexpressed, and the level of expression of BCL-X_S remained unaltered. Whether the consistent level of expression of BCL-X_S reflected inability of AAP to influence its expression remains unknown. Unaltered expression of BCL-X_S in the near total absence of BCL-2 expression raises questions regarding the death promoting role of BCL-X_S in vivo. The precise role of modified form of BCL-X_L remains elusive. However, this study may have demonstrated for the first time drug-induced changes in the expression of anti-apoptotic gene BCL-X_L, and a positive link between AAP-induced apoptotic death and modification of BCL-X_L protein in vivo.