Sex differences in the phase I (cytochrome P-450-catalyzed hydroxylations) and phase II (conjugations) metabolism of N-2-fluorenylacetamide (2-FAA) by the livers of 50-day-old Sprague-Dawley rats and effects of gonadectomy were determined. The higher level (1.4 times) of cytochrome P-450 in the microsomes of male rats correlated with their 8 and 1.3 times greater capacities to form 9-hydroxy(OH)-2-FAA and 7-OH-2-FAA respectively. One week after gonadectomy, the formation of 9-OH-2-FAA, the major metabolite in the male, was decreased by 70%, whereas in the female it was increased 1.3 times. Treatment of male rats with beta-naphthoflavone (beta-NF) increased the formation of phenolic metabolites and N-OH-2-FAA, but decreased that of 9-OH-2-FAA. The amounts of 9-OH-2-FAA were increased, however, in beta-NF-treated female and gonadectomized male rats. These sex hormone- and beta-NF-mediated differences in the extent of 9-hydroxylation of 2-FAA are discussed in relation to the fluctuations in the levels of specific cytochrome P-450 isozymes. In contrast to the phenolic metabolites and N-OH-2-FAA, 9-OH-2-FAA was a poor substrate for UDP-glucuronyltransferase; this conjugation was not induced by treatment of male rats with beta-NF. Hence, in the presence of male hormones, relatively large amounts of 9-OH-2-FAA were formed and possibly retained in the liver. A role of this alcohol as a potential promoter in hepatocarcinogenesis by 2-FAA is suggested.