The crystal structure of 5'-nucleotidase (5'-NT) from E. coli, also known as UDP-sugar hydrolase, has been determined at 1.7 Å resolution. Two zinc ions are present in the active site, which is located in a cleft between two domains. The dimetal center and a catalytic Asp-His dyad are the main players in the catalytic mechanism. Structure-based sequence comparisons show that the structure also provides a model for animal 5'-NTs, which together with other ectonucleotidases terminate the action of nucleotides as extracellular signaling substances in the nervous system.