Both N-hydroxy-2-acetamidofluorene (N-OH-AAF) and the heterocyclic analogue, 2-(N-hydroxyacetamido)carbazole (N-OH-AAC), were shown to be mechanism-based irreversible inhibitors (suicide inhibitors) of partially purified rat hepatic N-acetyltransferase (NAT) activity. Although N-OH-AAC exhibited an apparent first-order inactivation rate constant (ki) that was 7-fold lower than that of N-OH-AAF, their relative ki/KD values indicate that N-OH-AAC was the more potent and efficient inactivator of transacetylase activity. Inactivation of NAT activity by these N-arylhydroxamic acids appeared to involve contributions by electrophiles that react with the enzyme subsequent to release from the active site and by electrophiles that remain complexed with the active site. The irreversible nature of the enzyme inactivation was demonstrated by the failure to recover transacetylase activity upon either extensive dialysis or gel filtration of preparations that had been subjected to incubation with N-OH-AAF or N-OH-AAC. The use of the nucleophile N-acetylmethionine to trap the electrophilic reactants formed in the transacetylase-catalyzed bioactivation process resulted in a lower rate and extent of formation of methylthio adducts with N-OH-AAC than with N-OH-AAF. The results of this study indicate that N-OH-AAF and N-OH-AAC have potential for use as tools in the investigation of rat hepatic transacetylases.