To determine how 5-fluoro-dUMP modifications may affect its specificity, 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP were compared as inhibitors of thymidylate synthases isolated from parental and FdUrd-resistant mouse leukemia L1210 cells, human and rat colon adenocarcinomas, regenerating rat liver and the tapeworm, Hymenolepis diminuta, differing in sensitivity to time- and N5,10-methylenetetrahydrofolate-dependent inactivation by 5-fluoro-dUMP (Ki values ranging from 10−9 to 10−7 M). Inactivation by 2-thio-5-fluoro-dUMP, relative to 5-fluoro-dUMP, was 5-20-fold weaker, with specificity for inactivation of different thymidylate synthases paralleling that of 5-fluoro-dUMP. By contrast, 4-thio-5-fluoro-dUMP showed very different specificity, being as potent an inactivator for some enzymes as 5-fluoro-dUMP, but 45-85-fold weaker for others. The results suggest that an interplay between substituents at C(4) and C(5) of the pyrimidine ring may affect the specificity of thymidylate synthase inactivation.