The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cells, with K(i) values of 32 microM and 55 microM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K(i) value was 10(3)-10(4)-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximately 10(-5)M) than FdUrd (IC50 approximately 10(-9)M), 2-thio-FdUrd(IC50 approximately 10(-7)M) or 4-thio-FdUrd (IC50 approximately 5x10(-8)M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine.