The inhibitory effect of methotrexate (MTX) on [ 14C] formate incorporation into RNA and protein in Ehrlich ascites tumor cells is decreased by pretreatment with fluoropyrimidines. When the basal rate of [ 3H] deoxyuridine (UdR) incorporation into DNA is decreased to 26, 64 and 95% by 5-fluorodeoxyuridine (FUdR), the inhibitory effect of MTX on [ 14C] formate incorporation into (a) RNA is decreased to 40, 19 and 3%, and (b) protein is decreased to 52, 25 and 7%. MTX alone, however, suppressed [ 14C] formate incorporation into RNA and protein 56 and 67%, respectively. Free intracellular MTX is required for maximal suppression of [ 14C] formate incorporation into RNA and protein after pretreatment of cells with FUdR. When high-affinity binding sites for MTX are > 95% saturated with MTX, but free or exchangeable MTX is not present, [ 14C] formate incorporation into RNA and protein is stimulated in the presence of FUdR. However, as the intracellular levels of MTX in excess of the tightly bound fraction is increased with 1 μM and 10 μM MTX, there is progressive inhibition of [ 14C] formate incorporation into RNA and protein in cells exposed to FUdR. Theses data support the concept that fluoropyrimidine-induced antagonism to MTX is related, in part, to levels of reduced folates available for purine and protein synthesis.