In comparison with the very limited or not detectable reactivity of the versatile carcinogen N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) (1-3) with proteins and nucleic acids (4-7), its esters have shown a strong nonenzymatic reactivity at pH 7 with these tissue constituents and certain of their components (4, 8-13). The sulfonium derivatives formed by reaction of esters of N-hydroxy-AAF with methionine decompose spontaneously to yield 1- and 3-methylmercapto-AAF (0-CH3S-AAF) (8, 11), and the same compounds can be liberated by alkali at room temperature from the liver protein of rats fed N-hydroxy-AAF (11, 14). Similarly, a product obtained by enzymatic digestion of liver RNA from rats administered N-hydroxy-AAF appears to be identical with N-(guanosin-8-yl)-AAF, formed by reaction of esters of N-hydroxy-AAF with guanosine in vitro (11, 14). These findings indicate that esters of N-hydroxy-AAF are involved in the formation in vivo of protein- and nucleic acid-bound derivatives of N-hydroxy-AAF in the rat (11, 15-17). In a search for enzymatic esterification of N-hydroxy-AAF we have noted the enzymatic activation of N-hydroxy-AAF with 3′-phosphoadenosine-5′-phosphosulfate (PAPS) by the soluble fraction of male rat liver for reaction with endogenous protein or added methionine, sRNA, or DNA. These studies confirm and extend the recent findings of King and Phillips (18) for sRNA, which appeared while our studies were being prepared for publication. In addition, we have found that the soluble fractions from the livers of female rats and four male rodent species resistant to hepatocarcinogenesis by N-hydroxy-AAF have lower capacities to transfer sulfate from PAPS to N-hydroxy-AAF than do the soluble fractions of male rat livers. Except for rabbit liver these levels parallel the susceptibility of the livers of these species to carcinogenesis by N-hydroxy-AAF. Materials and Methods. Animals. Adult animals were obtained from the following dealers: male and female albino rats (Holtzman Rat Co., Madison, Wis.); male guinea pigs (O'Brien Co., Madison, Wis.); male Syrian golden hamsters (Con Olson Co., Madison, Wis.); male New Zealand rabbits (Willard Voss, Madison, Wis.); and male albino mice (CD-1 stock, Charles River Breeding Laboratory, Wilmington, Mass.).