Slices of liver from CD rats were evaluated by autoradiography after exposure to N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) labeled in the acetyl moiety with tritium. These in vitro experiments showed that the incorporation of the acetyl group of N-hydroxy-AAF was a sulfate-dependent enzymatic process in livers from male rats. Livers from females, and hyperplastic nodules and carcinomas induced in the livers of males, were essentially incapable of carrying out this reaction. The results of these in vitro experiments were compared with those obtained with liver sections from a male CD rat exposed in vivo to the tritiated N-hydroxy-AAF. A close relationship between covalent binding and necrosis was demonstrated by the observation that the acetyl moiety of N-hydroxy-AAF was preferentially incorporated into the periportal area, the same area in which liver necrosis occurs after a single injection of the hydroxamic acid. These data offer a biochemical rationale for the relative sensitivities of these cells to the toxic effects of N-hydroxy-AAF and its parent amide. There were no differences in the incorporation of ring-labeled N-hydroxy-4-acetylaminobiphenyl with respect to the sex of the animal or to the morphological characteristics of the tissue. The inhibition of the formation of these adducts by paraoxon suggests that they resulted from the action of deacetylases in the endoplasmic reticulum.