Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependences of the inactivation rate on time and accompanied by differing inhibition parameters. Considering importance for chemotherapeutic drug resistance, a possibility was tested of thymidylate synthase inhibition to be affected by post-translational modification, e.g. phosphorylation, by comparing sensitivities to inhibition by each of two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparation, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered with each inhibitor studied both the pattern of the dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy -dCMP was found strongly dependent on [Mg2+], the cations demonstrated previously to influence also activity of endogenous mouse TS isolated from tumour cells.