A sensitive immunofluorescence assay was developed for localizing acetaminophen (APAP) protein adducts in liver sections from treated mice. Affinity-purified anti-APAP antibodies, when applied to liver sections from mice given 600 mg APAP/kg, po, were preferentially localized in cells of the centrilobular region. At 30 min after dosing, covalently bound APAP was detected only in those cells most proximal to the central vein. Thereafter, binding spread throughout the centrilobular zone. However, by 8 hr the overall intensity of staining decreased and binding appeared more diffuse. Western blot analysis of electrophoretically resolved proteins from similarly treated mice revealed a corresponding temporal arylation of cytosolic proteins by APAP and indicated that the fluorescence detected at 30 min was associated with arylation of protein(s) of 44 kDa. The findings demonstrate the sensitivity and utility of immunohistochemical techniques in the study of covalently bound toxicants and emphasizes the temporal link between selective protein arylation in individually targetted cells to the development of APAP hepatotoxicity.