Studies of plasma from mice, rats, and human volunteers evaluated methods for the extraction and quantification of the positron emission tomography ligand [(18)F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([(18)F]CPFPX) and identification of its metabolites in plasma by thin-layer chromatography and high-performance liquid chromatography (HPLC). Analysis of human, mouse, and rat plasma extracts by HPLC identified four identical radioactive metabolites in each species. The low mass of radioligand administered to humans (0.5 - 5 nmol) prevented direct identification of metabolites. However, incubating liver microsomes with CPFPX and analysis by means of liquid chromatography-mass spectrometry (LC-MS) identified seven compounds, four having the same retention times as the metabolites in human plasma. Analysis of microsomal metabolites by LC-MS identified five [M + H](+) ions of m/z equivalent to hydroxy derivatives, 339, one of m/z equivalent to an oxo derivative, m/z 337, and one of m/z equivalent to a difunctionalized oxo-desaturation species, m/z 335, which is prominent in rat and mouse plasma and is the main metabolite in human plasma. An [M + H](+) ion corresponding to a N-dealkylated derivative was not detected. Thus, like the natural methylxanthines, CPFPX seems to undergo oxidation by liver microsomes but, unlike those methylxanthines, dealkylation did not occur. LC-MS experiments with "in source" fragmentation identified the cyclopentyl moiety to be the most functionalized part of the molecule by liver microsomes and in vivo oxidations. Except for two metabolites, hydroxylated at the N1 propyl chain, all oxidative modifications found took place at the cyclopentyl ring.