5,10-Dideazatetrahydrofolic acid (DDATHF) reduces de nova purine biosynthesis by inhibiting glycinamide ribonucleotide transformylase. ICI D1964 and CB3717 are folate-based inhibitors of thymidylate synthase (TS). Fluorodeoxyuridine (FdUrd) following metabolism to FdUMP also inhibits TS. In cultured L1210 cells DDATHF reduced the toxicity of ICI D1694, CB3717 and FdUrd in a concentration-dependent manner. This protection correlated with a prevention of the increase in intracellular dATP pools seen in cells exposed to the TS inhibitors alone. The possibility that DDATHF protection might be due to competition for cell entry is not likely since CB3717 and FdURD but not ICI D1694 enter the cell by independent transport processes. Exogenous hypoxanthine (HX) had no effect on the toxicity of TS inhibitors. However, HX increased the protective effect of DDATHF from ICI D1694 toxicity, had no effect on the CB3717-DDATHF interaction, and reduced the protective effect of DDATHF on FdUrd toxicity. HX prevented the fall in dATP levels caused by DDATHF addition to cells treated with TS inhibitors. HX had different effects on dTTP levels in cells treated with DDATHF and quinazoline TS inhibitors compared to FdUrd. Together these results support the hypothesis that imbalance in dTTP and dATP pools is an important determinant of cytotoxicity in antifolate-treated cells. In addition, these findings suggests that intracellular reduced folates interconvert to catalyse reactions in metabolically perturbed cells.