The crystal structure of the homodimer of quinone oxidoreductase fromEscherichia colihas been determined using the multiple isomorphous replacement method at 2.2 Å resolution and refined to an R-factor of 14.1%. The crystallographic asymmetric unit contains one functional dimer with the two subunits being related by a non-crystallographic 2-fold symmetry axis. The model consists of two polypeptide chains (residues 2 through 327), one NADPH molecule and one sulphate anion per subunit, and 432 water molecules. Each subunit consists of two domains: a catalytic domain and a nucleotide-binding domain with the NADPH co-factor bound in the cleft between domains. Quinone oxidoreductase has an unusual nucleotide-binding fingerprint motif consisting of the sequence AXXGXXG. The overall structure of quinone oxidoreductase shows strong structural homology to that of horse liver alcohol dehydrogenase. f2 f2Abbreviations used: QOR, quinone oxidoreductase; LADH, liver alcohol dehydrogenase; GDH, glucose dehydrogenase; MIR, multiple isomorphous replacement; SA, simulated annealing; NCS, non-crystallographic symmetry.