N-Hydroxy-2-acetylaminofluorene (NOH-2AAF) is converted by a rat liver 100,000 x g supernatant amidase to N-hydroxy-2-aminofluorene (NOH-2AF), a potent frameshift mutagen. Upon addition of sulfation cofactors, a cytosol sulfotransferase catalyzed the formation of the N-O-sulfate ester of NOH-2AAF, which is a less potent mutagen than NOH-2AF. Addition of DNA, RNA or purine and pyrimidine bases to mutagenesis assays did not alter the observed mutagenesis, although protein covalent binding of 14C (acetyl)-NOH-2AAF was diminished. Addition of ascorbic acid or reduced pyridine nucleotides while not affecting the generation of the N-O-sulfate ester decreased the observed protein covalent binding and increased the mutagenic response ten-fold while generating the nonmutagenic species 2AAF. These results are consistent with the proposal that a radical cation intermediate generated by ascorbic and reduced pyridine nucleotides is a potent mutagenic species. Further, the electrophilic species which reacts with protein, does not appear to be important for the mutagenesis of N-hydroxy-acetylarylamides.