1. 2-Acetylaminofluorene (AAF) was converted by rat liver microsomal and cytosolic enzymes to 2-aminofluorene (AF), 2-glycoloylaminofluorene (GAF), 2-acetylaminofluoren-3-, -7-, and -9-ol (3-, 7-, 9-hydroxy-AAF), and 2-acetylaminofluoren-9-one (AAF-9-one). In addition, a new metabolite MX1was detected.2. AAF was converted by rabbit liver microsomal and cytosolic enzymes toN-hydroxy-AAF, GAF, 5-, 7-, and 9-hydroxy-AAF, AAF-9-one, 5- and 7-hydroxy-AAF-9-one (new compounds), and AF, indicating species differences in theN- and ring-hydroxylation of AAF and secondary oxygenation of AAF. In addition, an unknown metabolite MX2was detected.3. AAF-9-one was converted by rat liver microsomal and cytosolic enzymes to optically active 9-hydroxy-AAF and 7-hydroxy-AAF-9-one; in addition MX1was found.4. Rabbit liver microsomal and cytosolic enzymes converted AAF-9-one to 2-aminofluoren-9-one (AF-9-one), 9-hydroxy-AAF,N-hydroxy-AAF-9-one, GAF-9-one, 7-hydroxy-AAF-9-one, and 7,9-dihydroxy-AAF. In addition, metabolite MX1and its dihydro-dihydroxy derivative were found.5. These results indicate that AAF and AAF-9-one have common metabolic pathways, as AAF after primary oxygenation to 9-hydroxy-AAF and partial dehydrogenation to AAF-9-one, undergoes secondary oxygenation to 7-hydroxy-AAF-one and MX1as well as the corresponding dihydro-dihydroxy derivatives.