The interactions of thymidylate synthase (TS) with deoxyuridylate (dUMP), deoxythymidylate (dTMP) and 5-fluorodeoxyuridylate (FdUMP) were examined by 31P-NMR. Single 31P resonances appeared at 3.3 ppm, 3.2 ppm and 3.0 ppm from the standard, 85% phosphoric acid, for unbound dUMP, dTMP, and FdUMP, respectively. Incubation of the enzyme with either dUMP or dTMP, alone, resulted in new resonances at 3.9 and 3.6 ppm, respectively, which were assigned to noncovalent complexes with the enzyme. The same experiment employing FdUMP as the ligand gave two new resonances appearing at 3.6 and 4.6 ppm, which were attributed to noncovalent and covalent binary complexes, respectively. When the cofactor, CH2H4 folate, was present in the solution with enzyme and FdUMP, a new resonance appeared at 5.1 ppm, corresponding to the covalent inhibitory ternary complex. The ternary complex comprised of the enzyme, dUMP and the quinazoline folate CB 3731 produced a resonance at 5.0 ppm at the expense of the resonance due to the enzyme-dUMP binary complex at 3.9 ppm. Similarly, the ternary complex consisting of TS with dTMP and CB 3731 showed a deshielding of the resonance at 3.6 ppm by 0.8 ppm. A maximum binding of 1.5 nucleotides per enzyme dimer was found for dUMP and dTMP in both the presence and the absence of the quinazoline folate. The deshielding observed was attributed to changes in the interaction of the phosphate group with the nearby residues of the active site of the enzyme.