5-Fluorodeoxyuridine inhibited DNA synthesis of mitogen-stimulated T and B cells as determined by either incorporation of 3H-thymidine to the cells or measure of DNA content in the cells by microdensitometry. 5-Fluorodeoxyuridine concentrations necessary to inhibit DNA synthesis by 50% in the stimulated cultures were high (2 micrograms/ml and 7 micrograms/ml for B and T cells, respectively). Addition of exogenous thymidine (100 micrograms/ml) partially reversed the inhibition of DNA synthesis by 5-fluorodeoxyuridine (10 micrograms/ml). These data indicate that lymphocytes possess a mechanism of resistance to inhibition of thymidilate synthetase by 5-fluorodeoxyuridine. On the other hand, the inhibition of mitogen-induced blastogenesis is not only mediated by an effect on thymidilate synthetase but could also be mediated by other mechanisms, such as inhibition of nucleic acid precursor transport into the cells.