Acetaminophen (4'-hydroxyacetanilide), a widely used analgesic/antipyretic drug, is hepatotoxic in large doses, whereas the m-hydroxy isomer of acetaminophen, 3'-hydroxyacetanilide, is not hepatotoxic. Both are oxidized by mouse liver cytochromes P-450 to reactive metabolites that bind covalently to hepatic proteins. Because previous studies have shown that peak levels of liver protein adducts formed after the administration of each of these compounds to mice are nearly equivalent, and because liver protein adduct formation correlates with hepatotoxicity caused by acetaminophen in mice, we investigated the abundance and patterns of protein adducts formed by acetaminophen and its regioisomer for significant differences. Hepatotoxic doses of acetaminophen to mice significantly altered the abundances of several liver proteins 2 h after dosing as revealed by densitometric analysis of two-dimensional electrophoretic patterns of these proteins. The same analysis after the administration to mice of 3'-hydroxyacetanilide indicated that this nonhepatotoxic regioisomer of acetaminophen caused several similar changes in protein patterns, but also revealed some significant differences. Binding of radiolabeled acetaminophen and 3'-hydroxyacetanilide to hepatic proteins corroborated and extended these results. Two hours after the administration of 14C-labeled analogs of these two compounds to mice, at a time when their extent of total covalent binding to hepatic proteins is approximately equivalent, there are many similarities but also some differences in selectivity of proteins that are adducted, as revealed by both one-dimensional and two-dimensional gel electrophoresis followed by phosphorimage analysis of radiolabel bound to protein bands. Moreover, protein adducts formed from 3'-hydroxyacetanilide were found to be less stable than those formed from acetaminophen under the conditions of electrophoretic analysis. Furthermore, a comparison of radiodetection and immunodetection of protein adducts formed from acetaminophen with an antibody specific for acetaminophen protein adducts indicates that the antibody detects most of the same proteins that are radiolabeled and that the relative quantitative contribution of various adducts to the overall covalent binding of acetaminophen to proteins is approximately the same by both methods. Thus, 3'-hydroxyacetanilide should prove to be a useful tool to aid in the discrimination of hepatic acetaminophen protein adducts that may be critical or noncritical to survival of hepatocytes.