Overdose of acetaminophen (APAP) causes acute hepatotoxicity in rodents and man. The mechanism underlying APAP-induced liver injury remains unclear, but experimental evidence strongly suggests that activation of APAP and subsequent formation of protein adducts are involved in hepatotoxicity. Using proteomics technologies, we constructed a two-dimensional protein database for mouse liver, comprising 256 different gene products and investigated the proteins affected after APAP-induced hepatotoxicity. Adult male mice received a single dose of APAP (100 or 300 mg/kg) or its nontoxic regioisomer 3-acetamidophenol (AMAP, 300 mg/kg). The extent of liver damage was assessed 8 h after administration by increased liver enzyme release and histopathology. Changes in the protein level were studied by comparison of the intensities of the corresponding spots on two-dimensional (2-D) gels. The expression level of about 35 of the identified proteins was modified due to treatment with APAP or AMAP. The observed changes were usually in the order of 10-50% of the control value and were more marked in the high- than in the low-dose of APAP-treated animals. Most of the changes caused by AMAP occurred in a subset of the proteins modified by APAP. Many of the proteins showing changed expression levels are either known targets for covalent modification by N-acetyl-p-benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP-induced hepatotoxicity.