The in vitro metabolism of 1-(4-[(18)F]fluoromethylbenzyl)-4-phenylpiperazine ([(18)F]1) and 1-(4-[(18)F]fluorobenzyl)-4-phenylpiperazine ([(18)F]2) was investigated using mouse liver S9 fraction. Results were compared to those of in vivo metabolism using mouse blood and bone and to in vitro metabolism using mouse liver microsomes. Defluorination was the main metabolic pathway for [(18)F]1 in vitro and in vivo. Based on TLC, HPLC and LC-MS data, [(18)F]fluoride ion and less polar radioactive metabolites derived from aromatic ring oxidation were detected in vitro, and the latter metabolites were rapidly converted into the former with time, whereas only the [(18)F]fluoride ion was detected in vivo. Similarly, the in vitro metabolism of [(18)F]2 using either S9 fraction or microsomes showed the same pattern as the in vivo method using blood; however, the radioactive metabolites derived from aromatic ring oxidation were not detected in vivo. These results demonstrate that liver S9 fraction can be widely used to investigate the intermediate radioactive metabolites and to predict the in vivo metabolism of radiotracers.