Identification of proteins that covalently bind acetaminophen (APAP) is essential for a clearer understanding of the hepatotoxicity that results after an APAP overdose. Birge et al. (1991) have reported that in our mouse model system a membrane-associated 44-kDa acetaminophen-binding protein is the earliest target detected immunochemically following administration of an hepatoxic dose of APAP. To identify this 44-kDa protein, liver microsomes from mice administered 600 mg APAP/kg were extracted with Triton X-114 and the resulting aqueous fraction was adsorbed to DEAE-cellulose. Further purification of the DEAE eluate by reverse-phase HPLC and by two-dimensional (2D) gel electrophoresis indicated that four proteins of approximately 44 kDa with pIs ranging from 6.7-7.0 were targeted by APAP. The most highly arylated of these 44-kDa isoforms (pI 7.0) was excised from 2D gels, digested with trypsin, and the resulting peptides were separated by reverse-phase HPLC. The two best resolved peptides were sequenced and 14 of the 15 amino acids detected were found to be identical to subunits of mouse liver glutamine synthetase (EC 6.3.1.2). Purification of glutamine synthetase from APAP-treated mice confirmed that the enzyme is indeed targeted by APAP in vivo. Since the intracellular activity of glutamine synthetase is significantly decreased after the administration of APAP to hepatocytes in culture, it is likely that the arylation of this enzyme may be involved in the ensuing hepatotoxicity.