© 2017 American Chemical Society. A novel method for radiolabeling perfluorinated alkyl substances (PFAS) with fluorine 18 has been developed, and after purification, the stability and biological distribution in healthy mice were evaluated. Three PFAS, [18F]PFOA (C8), [18F]PFHxA (C6), and [18F]PFBA (C4), were readily labeled and isolated in average yields between 12 and 31%. The stability of each compound was monitored in 0.1% ammonium hydroxide (NH4OH) in methanol, in saline, and in human, mouse, and rat sera. The amount of intact, radiolabeled PFAS was determined by radiometric instant thin layer chromatography and was calculated by the amount of free fluorine 18 observed over time. All compounds were highly stable in 0.1% NH4OH in methanol and saline, with <10% defluorination observed after 4 h. Interestingly, each compound had differing affinities for the serum proteins. In vivo biodistribution studies in mice showed uptake in all organs examined, with the highest uptake being exhibited in the liver for both [18F]PFOA and [18F]PFHxA and the stomach for [18F]PFBA. The results of this initial study suggest that this method could be valuable in helping to determine the biological uptake of any PFAS in mammals.