When hepatotoxic doses of [ring-U-14C]acetaminophen ([ring-U-14C]APAP) were administered to mice, radioactivity became bound irreversibly to hemoglobin as well as to proteins in the liver and kidney. The covalent binding to hemoglobin was dose-dependent, and in phenobarbital-pretreated mice occurred to the extent of approximately 8% of the corresponding binding to liver proteins. Degradation of the modified globin by acid hydrolysis yielded 3-cystein-S-yl-4-hydroxyacetanilide as the major radioactive product, accounting for approximately 70% of protein-bound drug residues. This finding is consistent with the view that the majority of covalent binding of APAP to proteins is mediated by N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which preferentially arylates cysteinyl thiol residues. However, after administration of [acetyl-3H]APAP to mice, it was found that approximately 20% of the drug bound to hemoglobin had lost the N-acetyl side-chain, indicating the existence of a second type of APAP-protein adduct. One minor component of the globin hydrolysate was identified as S-(2,5-dihydroxyphenyl)-cysteine, which most likely arises from binding to hemoglobin of p-benzoquinone, a hydrolysis product of NAPQI. The two adducts reported represent the first identified examples of arylating drugs binding to hemoglobin. Experiments on the influence of different cytochrome P-450 inducing agents on the ratio of drug bound to hemoglobin versus hepatic proteins suggested that the reactive metabolites of APAP are formed in the liver and migrate to the erythrocyte, rather than being produced by hemoglobin-catalyzed oxidation of APAP. These findings imply that the reactive metabolites of APAP escape from hepatocytes in some latent forms, which then participate in the arylation of protein thiols in red blood cells and, possibly, at other remote sites.