Murine P388 cells incubated in vitro with the anticancer drug arabinosyl 2-fluoroadenine accumulate its 5'-triphosphate, F-araATP, as the major phosphorylated metabolite. A new chromatographically separate metabolite that accumulated to levels 10% of that of F-araATP was identified as 2-fluoro-ATP, by the following criteria. 1. The metabolite coeluted with the authentic compound on anion-exchange HPLC. 2. Dephosphorylation of the metabolite yielded a compound that was chromatographically identical to 2-fluoroadenosine. 3. The compound was sensitive to NaIO4 oxidation. Cellular incubation experiments indicated that 2-fluoroadenine, but not arabinosyl 2-fluorohypoxanthine, was the likely intermediate in the formation of 2-fluoro-ATP.