The cytotoxicity of 5-fluorouridine (FUrd) results from actions directed at the synthesis of both DNA and RNA. The role of mRNA as a target for FUrd was investigated by selectively decreasing the incorporation of FUrd into RNA polymerase II transcripts of K-562 erythroleukemia cells, which was accomplished by the addition of alpha-amanitin to cultures of K-562 cells permeabilized with lysolecithin. In these cells alpha-amanitin at concentrations of 1-5 micrograms/ml inhibited the incorporation of [3H]-uridine into polyadenylated RNA by up to 45% and decreased the steady-state levels of two specific mRNAs but had no effect on poly A- RNA synthesis. alpha-Amanitin decreased the incorporation of FUrd into poly A+ RNA by up to 60%. The decrease in FUrd incorporation produced by alpha-amanitin was accompanied by an antagonism of the growth inhibitory effects of the fluorinated pyrimidine nucleoside by the mycotoxin, as measured by both growth in suspension culture and colony formation in 0.12% agar. Antagonism between these agents increased as the concentration of alpha-amanitin was elevated; furthermore, it was sequence-dependent, occurring only when alpha-amanitin preceded FUrd. These findings provide evidence that the actions of FUrd directed against mRNA are antagonized when FUrd incorporation into mRNA transcripts is decreased and that the effects of FUrd on mRNA produce cytotoxic consequences.