The effects of 5-trifluoromethyluracil (F3Thy), 5-trifluoromethyl-2'-deoxyuridine (F3dThd), F3dThd-5'-P, and F3dThd-5'-methylphosphonate on the growth of HeLa cells, Novikoff hepatoma cells, L5178Y mouse leukemia cells, and on the replication of vaccinia virus in HeLa cells have been determined. F3Thy and F3dThd-5'-methylophosphonate were approximately 500-fold and 100-fold less effective, respectively, than F3dThd or F3dThd-5'-P in their inhibition of these cells. F3dThd and F3dThd-5'-P are potent inhibitors of vaccinia viral replication of HeLa cells. The nucleoside and nucleotide were 1,000-fold more inhibitory than the free base. F3dThd was nost inhibitory when added between 1 and 2 h post-infection; however, it was also somewhat inhibitory when added at later times.