Crystals of recombinant Pseudomonas putida catechol 2,3-dioxygenase, metapyrocate-chase, composed of four identical subunits, each with a molecular mass of 35 kDa and one nonheme ferrous iron, have been grown by the vapor diffusion method using sodium citrate as the precipitant. Repeated macroseeding and the addition of ethanol to protein solutions were together effective for obtaining crystals suitable for further crystallographic characterization. The crystals belong to the tetragonal space group P4(2)2(1)2 with unit-cell dimensions of a = b = 266 A, c = 60 A. They diffracted beyond 2.5 A resolution with synchrotron radiation. Assuming that one tetramer (alpha-Fe2+)4 is contained in an asymmetric unit, the crystal volume per unit molecular mass, Vm, is calculated to be 3.8 A3/Da, which corresponds to the solvent content of 67.6%.