In vitro metabolism of acetylcholinesterase inhibitors containing 3-[(18)F]fluoromethylbenzyl- ([(18)F]1) and 4-[(18)F]fluorobenzyl-piperidine moieties ([(18)F]2) was studied and compared with the in vivo metabolism. Defluorination of the [(18)F]1 mainly occurred to generate [(18)F]fluoride ion both in vitro and in vivo. In contrast, the [(18)F]2 was converted into an unknown polar metabolite in both metabolism methods and another metabolite, 4-[(18)F]fluorobenzoic acid in vitro. These results demonstrated that the in vitro method can be used to predict the in vivo metabolism of both radiotracers.