Traditional fluorine-19 nuclear magnetic resonance (19F NMR) studies of thymidylate synthase (TS) have utilized the fluorine substituent of 5-fluorodeoxyuridine 5'-monophosphate (FdUMP), a mechanism-based inhibitor of the enzyme, in complexes with various folate and folate analogues in order to establish a paradigm for the formation of binary and ternary complexes. In order to extent and confirm this paradigm, complexes of thymidylate synthase (TS) and the N-10-(fluoroethyl)quinazolinylfolate analogue CB3731 with either deoxyuridine 5'-monophosphate (dUMP), deoxythymidine 5'-monophosphate (dTMP), or FdUMP were examined from the perspective of the folate analogue using 19F NMR. The spectrum of the free folate analogue gave rise to a multiplet centered at -57.0 ppm, which was broadened by approximately 50% upon incubation with the enzyme. The use of FdUMP with CB3731 afforded us the opportunity to compare the spectrum obtained for the folate with that of the nucleotide. This comparison led to the assignment of the resonance at -53.5 ppm as representing the noncovalent TS:FdUMP:CB3731 ternary complex, while a new resonance at -52.0 ppm corresponded to the species in which the nucleotide is covalently attached to the enzyme and the folate is noncovalently associated. Ternary complexes consisting of TS, CB3731, and either dUMP or dTMP displayed a resonance at -53.5 ppm which represented the noncovalent TS-nucleotide adduct. None of the TS:nucleotide:CB3731 ternary complexes, however, was stable to native polyacrylamide gel electrophoresis.