The enzyme dUTPase catalyses the hydrolysis of dUTP and maintains a low intracellular concentration of dUTP so that uracil cannot be incorporated into DNA. dUTPase from Escherichia coli is strictly specific for its dUTP substrate, the active site discriminating between nucleotides with respect to the sugar moiety as well as the pyrimidine base. Here we report the three-dimensional structure of E. coli dUTPase determined by X-ray crystallography at a resolution of 1.9 A. The enzyme is a symmetrical trimer, and of the 152 amino acid residues in the subunit, the first 136 are visible in the crystal structure. The tertiary structure resembles a jelly-roll fold and does not show the 'classical' nucleotide-binding domain. In the quaternary structure there is a complex interaction between the subunits that may be important in catalysis. This possibility is supported by the location of conserved elements in the sequence.