Abstract Rat-liver rRNA and DNA were isolated 16 h after intraperitoneal injection of [9- 14C] N-hydroxy-2-[2'- 3H]acetylammofluorene. The N-acetyl group was retained on most of the fluorene moieties bound to the rRNA (75% bound wilh the acetyl group on them; 25% without the acetyl group), whereas, a larger fraction of the N-acetyl groups was lost on the fluorene moieties bound to the DNA (35% bound with acetyl group; 65% without acetyl). The products of the reaction of [9- 14C]sodium ( N-[2'- 3H]acetyl- N-2-fluorenylhydroxylamine β- d-glucosid)uronate with yeast tRNA and calf-thymus DNA in vitro at pH 7.4 at 37° contained only a minor fraction (20–35%) of bound fluorene moieties with the N-acetyl group. The loss of the N-acetyl group during the course of this reaction was pH-dependent. The data presented are consistent with the hypothesis that the glucuionide of N-hydroxy-2-acetylaminofluorene accounts for most of the binding of the carcinogen N-hydroxy-2-acetylaminofluorene to rat-liver DNA in vivo.