A new method was devised to study adduct formation of N-acetyl-L-methionine (N-Ac-met) with activated species of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF). Following degradation of the adducts, the isomeric methylthio derivatives of 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) were separated and quantified by h.p.l.c. With the use of this method the nucleophilicity of N-Ac-met toward enzymatically produced N-acetoxy and N-sulfonoxy derivatives of 2-AF and 2-AAF, respectively, and toward the synthetic model ester, N-acetoxy-2-acetylaminofluorene (N-OAc-2-AAF), was determined. For comparison, the interaction of tRNA and polyguanylic acid (poly G) with the above compounds was measured by standard procedures. tRNA was an efficient acceptor of enzymatically formed N-acetoxy and N-sulfonoxy derivatives as well as of N-OAc-2-AAF. The nucleophilicity of N-Ac-met toward enzymatically formed N-sulfonoxy-2-AAF was comparable with that of tRNA. However, its reactivity with N-OAc-2-AAF and enzymatically generated N-acetoxy-2-aminofluorene (N-OAc-2-AF) was only 20% of that of tRNA. Poly G was as reactive as tRNA toward synthetic N-OAc-2-AAF and enzymatically generated N-OAc-2-AF but was only 25% as efficient as tRNA or N-Ac-met in accepting enzymatically formed N-sulfonoxy-2-AAF. The difference in the interaction of the three nucleophiles with N-OAc-2-AAF and N-OAc-2-AF compared with N-sulfonoxy-2-AAF indicate that N-OAc-2-AAF is not a general model of the ultimate electrophilic metabolites of 2-AAF. Studies of the kinetics of the interaction of N-OAc-2-AAF with N-Ac-met, tRNA and poly G demonstrated dependence of adduct formation on nucleophile concentration, indicative of a bimolecular mechanism. Arylamidonium or nitrenium ions are therefore not necessarily the ultimate electrophilic metabolites of 2-AAF obligatory for interaction with cellular nucleophiles. There was no evidence that nitrenium ions are intermediates in the cytosolic reduction of N-OH-2-AAF to 2-AAF.