The purpose of this study was to compare the pools of free FdUMP derived from 5-fluorouracil (FUra) and of dUMP synthesized de novo in Hep-2 and S-180 cells, their relationship to inhibition of thymidylate synthase (dTMP synthase; EC 2.1.1.45), and the effect of excess folinic acid (CF) on these parameters. These cells differ 50-fold in their sensitivity to FUra and, in the absence of thymidine, dTMP synthase is the growth-limiting site of action of FUra in S-180 cells, but in Hep-2 cells this site becomes growth-limiting only in the presence of excess folates. In both cells after a 3-hr incubation with varied concentrations of FUra, FdUMP comprised only 0.1-0.2% of the total acid-soluble pools derived from FUra. The changes in dUMP and FdUMP pools paralleled each other, dUMP being 1000-2000 times higher than FdUMP. The pools of dUMP increased only when dTMP synthase was significantly inhibited. This occurred in S-180 cells above 3 microM FUra and in Hep-2 cells above 30 microM, where the residual dTMP synthase was similar in both cells. Under these conditions, the dUMP and FdUMP pools in Hep-2 cells were 2 and 4 times higher, respectively, than in S-180 cells. After FUra removal, both pools continued to increase, dUMP and FdUMP pools in Hep-2 cells rising 6-fold and 10-fold higher, respectively, than in S-180 cells. The dTMP synthase inhibition and the high nucleotide pools in Hep-2 were short-lived, whereas in S-180 cells the inhibition and the pools were maintained longer. Excess CF retarded the recovery of dTMP synthase after FUra removal only in Hep-2 cells and led to a further increase in dUMP and FdUMP pools in these cells, while having no effect in S-180 cells. These data indicate that a high capacity of cells to accumulate free FdUMP does not alone guarantee that dTMP synthase inhibition will be growth-limiting. The relationship shown here between excess CF, dTMP synthase recovery, and the nucleotide pools suggests that some cell types, such as Hep-2, in spite of high levels of FdUMP, require in addition an excess of folates to retard dTMP synthase recovery and make it growth-limiting.