Free energy perturbation calculations have been applied to evaluate the relative free energies of binding of 2'-deoxyuridine-5'-monophosphate (dUMP) and its 2- and/or 4-thio and/or 5-fluoro analogues to the wild-type E. coli thymidylate synthase (ecTS). The results accurately reproduce experimentally measured differences in the free energy of binding of dUMP versus 5-fluoro-dUMP to thymidylate synthase. They indicate that preferred binding of dUMP compared to 5-fluoro-dUMP in the binary complex is equally related to (i) more favorable electrostatic interactions of the dUMP molecule in the enzyme active site, and (ii) its less favorable solvation in the aqueous solution. The relative free energies of binding in the binary complex show moderate and qualitatively indistinguishable discrimination among the studied fluorinated and non-fluorinated 2- and/or 4-thio analogues of dUMP. The binding free energies of monothio analogues of dUMP and 5-fluoro-dUMP correspond quite well with experimentally measured activities of these nucleotides in the thymidylate synthase reaction. On the other hand, the binding free energies of both dithio analogues, 2,4-dithio-dUMP and 2,4-dithio-FdUMP, show lack of such correlation. The latter suggests that very low activities of the dithio analogues of dUMP and 5-fluoro-dUMP may relate more to the covalent reaction of these nucleotides within the ternary complex with TS and 5,10-methylenetetrahydrofolate, than to their pre-covalent binding. We speculate that a lack of substrate activity of 2,4-dithio-dUMP is related to the high aromaticity of its pyrimidine ring that prevents the Michael addition of the active site cysteine thiol to the pyrimidine C6 atom. A stronger affinity of the fluorinated analogues of dUMP to thymidylate synthase, compared to the non-fluorinated congeners, results from the fluorine substituent producing a local strain in the C6 region in the pyrimidine ring, thus sensitizing C6 to the Michael addition of the cysteine thiol.