Results are described which demonstrate that the cytotoxic action of 2',5-difluoro-1-arabinosyluracil (FFara-Ura) involves conversion to the corresponding 5'-phosphate, FFara-UMP, and subsequent inhibition of thymidylate synthetase. The evidence for this is as follows: (a) cells lacking thymidine kinase are 120-fold more resistant to FFara-Ura; (b) FFara-Ura markedly inhibits the incorporation of 2'-deoxyuridine (dUrd) into DNA with little or no effect on 2'-deoxythymidine (dThd) incorporation; (c) FFara-Ura causes changes in deoxynucleoside triphosphate pool sizes, which are characteristic of specific inhibition of dTMP synthetase. Binding and spectroscopic studies demonstrate that FFara-UMP inactivates dTMP synthetase from Lactobacillus casei in a manner analogous to that described for FdUMP. Furthermore, FFara-Ura is not a substrate for the pyrimidine phosphorylases; the significance of this finding with regard to the possible chemotherapeutic utility of FFara-Ura is discussed.