The N-hydroxylation of N-3-fluorenylacetamide (3-FAA), an isomer of the carcinogen, N-2-fluorenylacetamide (2-FAA), by hepatic microsomes of untreated and 3-methylcholanthrene (3-MC)-treated guinea pigs was found to be of a similar low order as that previously observed in the rat. Hepatic microsomes of the guinea pig and of the rat converted 3-FAA to N-(9-hydroxy)-3-FAA and to N-(9-oxo)-3-FAA. These new metabolites were separated and identified by high-pressure liquid chromatography (h.p.l.c.). N-(9-hydroxy)-3-FAA was the major product of the hydroxylation of 3-FAA by hepatic microsomes of the rat or guinea pig exceeding the formation of N-(7-hydroxy)-3-FAA, the principal phenolic metabolite of 3-FAA. The amounts of N-(9-oxo)-3-FAA formed were about one-third of the amounts of N-(9-hydroxy)-3-FAA produced. In contrast to the formation of phenolic metabolites, the hydroxylation of 3-FAA to N-(9-hydroxy)-3-FAA was not increased by pretreatment of guinea pigs or rats with 3-MC. Similarly, pretreatment of rats with PB did not enhance the yield of N-(9-hydroxy)-3-FAA. Co inhibited the formation of N-(9-hydroxy)-3-FAA by 80 per cent. These data lead us to conclude that the formation of N-(9-hydroxy)-3-FAA is catalyzed by a microsomal hemoprotein not identical with cytochrome P1-450 or P-450. In contrast to 3-FAA, 2-FAA appeared to be a poor substrate for hydroxylation to the N-(9-hydroxy)-2-FAA. The susceptibility of 3-FAA to hydroxylation at carbon-atom 9 of the fluorene moiety may be rationalized by resonance structures in which carbon-atom 9 is positively charged and acts as a electrophilic center. Similar resonance structures cannot be written for 2-FAA.