1H-3-Hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 catalyses the oxygenolysis of 1H-3-hydroxy-4-oxoquinoline to form N-formylanthranilic acid and carbon monoxide without the aid of cofactors. Both N-terminally His6-tagged and native QDO were overexpressed in Escherichia coli and purified by conventional chromatographic procedures. Untagged QDO, but not His6-tagged QDO, was crystallized by the vapour-diffusion method, giving hexagonal bipyramid crystals belonging to space group P6122. Selenomethionine-containing native QDO was prepared and crystallized under identical conditions. The unit-cell parameters were a = b = 90.1, c = 168.6 Å, α = β = 90, γ = 120°. Using synchrotron radiation, these crystals diffract to 2.5 Å. The expression, purification and crystallization of QDO are reported here.