Ecotin, a novel serine protease inhibitor isolated from Escherichia coli, has been crystallized using polyethylene glycol 1500 as the precipitating agent. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters of a = 39.22 A, b = 84.86 A, and c = 98.74 A. The asymmetric unit contains one dimeric molecule of ecotin, with a crystal volume per protein mass (Vm) of 2.55 A3/Da and a solvent content of 51.8% by volume. The crystals diffract to at least 2.2 A using a conventional X-ray source, and X-ray data have been collected to 2.7 A Bragg spacing from a native crystal.