The mutagenic activations of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by subcellular fractions of the livers of the Sprague-Dawley rat, C57BL mouse, Hartley guinea pig, Syrian golden hamster, and Macaca fuscata fuscata monkey were examined for sensitivity to paraoxon, which inhibits a deacetylase but not an arylhydroxamic acid acyltransferase. The mutagenic activation by liver microsomes was almost entirely mediated by a paraoxon-sensitive enzyme in all the animals tested. In contrast, the mutagenic activation by liver cytosol was mediated mostly by a paraoxon-sensitive enzyme in mice and guinea pigs, mostly by a paraoxon-resistant enzyme in rats and hamsters, and by both enzyme types in the monkey. In rats and guinea pigs, attempts were made to identify the enzymes causing mutagenic activation in the liver cytosol by a comparison of the elution positions of these enzymes in gel filtration and DEAE-cellulose column chromatography with those of known enzymes. In the rat liver cytosol, the mutagenic activation was mediated not only by acyltransferase but also by an unknown enzyme, which was resistant to paraoxon and differed from acyltransferase in chromatographic behavior. In the guinea pig liver cytosol, the activation was due to deacetylase activities, which could be separated into four fractions by gel filtration. These data indicate that species differ in the kinds of liver cytosolic enzymes involved in mutagenic activation of N-OH-2-FAA but not in the kind of liver microsomal enzyme involved.