The complexes that thymidylate synthetase (TSase) forms with various potent inhibitors have been intensively studied and thoroughly reviewed. Of particular significance is the covalent ternary complex of TSase-FdUMP-5,10-CH2H4PteGlu. FdUMP is the active metabolite of the widely used anti-cancer drug 5-fluorouracil. This complex is thought to be analogous to a steady-state intermediate of the normal enzyme reaction with the substrate dUMP. In this review, we examine the properties of TSase-dUMP complexes in order to determine if there is an experimental basis for drawing a close analogy between dUMP and FdUMP in their interaction with TSase, and also to evaluate data indicating a potential chemotherapeutic value for TSase-dUMP complexes formed in the presence of folate analogs.