Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.