The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on RNA metabolism was evaluated both in whole cells and in cell-free systems. F-ara-A was converted to its 5'-triphosphate, F-ara-ATP, in cells and then incorporated into RNA as well as DNA. F-ara-A inhibited RNA synthesis in cultured cells in a concentration-dependent manner. This inhibition was mediated mainly by F-ara-ATP. Experiments using isolated nuclei demonstrated that RNA polymerases I, II, and III accounted for 24, 73, and 3% of the total RNA synthesis activity, respectively. About 88% of the total inhibition was attributed to the suppression of RNA polymerase II activity. In cultured cells, F-ara-A was preferentially incorporated into the poly(A)+ RNA fraction. Approximately 78% of the incorporated F-ara-A monophosphate residues were located at the terminal position of the RNA chain. The incorporation of F-ara-A monophosphate into mRNA resulted in premature termination of the RNA transcript and impaired its functioning as a template for protein synthesis. The inhibitory action of F-ara-A on RNA metabolism is a unique property of this compound, differing from the action of arabinosylcytosine and arabinosyladenine.