The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 Å resolution using multiple-isomorphous-replacement techniques. The amino acid sequence deduced from the deoA DNA sequence is also reported. Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallographic 2-fold axis. Each subunit is composed of a small α-helical domain of six helices and a large α/β domain. The α/β domain includes a six-stranded mixed β-sheet and a four-stranded antiparallel β-sheet. The active site has been identified by difference Fourier analyses of the binding of thymine and thymidine and lies in a cavity between the small and large domains. The central β-sheet is splayed open to accommodate a putative phosphate-binding site which is probably occupied by a sulfate ion in the crystal.