The crystal structure of an Escherichia coli thymidylate synthase (TS) ternary complex containing 5-fluoro-2'-deoxyuridylate (FdUMP) and 10-propargyl-5,8-dideazafolate (PDDF) has been determined and refined at 2.3 A resolution. Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed beta-sheet. The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a beta-sandwich. In contrast to other proteins of known structure in which aligned beta-sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist. The substrate-binding pocket is a large funnel-shaped cleft extending some 25 A into the interior of each subunit and is surrounded by 30 amino acids, 28 from one subunit and two from the other. FdUMP binds at the bottom of this pocket covalently linked through C-6 to the sulfur of Cys146. Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF. The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoyl glutamate tail exposed at the entrance to the active site cleft. Ternary complex formation is associated with a large conformational change involving four residues at the protein's carboxy terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent.