The crystal structure of cleaved antithrombin III (ATIII) has been determined to 3.2 A resolution by single isomorphous replacement, real space density modification and phase extension protocols. The heavy-atom sites and the first molecular envelope were determined owing to the molecular replacement solution previously reported and partially refined. Refinement of the two molecules of the asymmetric unit led to a crystallographic R-factor of 0.212 for all reflections between 8.0 and 3.2 A, without inclusion of water molecules. The root-mean-square deviation from ideal values is, respectively, 0.015 A and 3.6 degrees for bond lengths and bond angles. The topology of the molecule closely resembles that of cleaved serpins inhibitors with the two residues forming the reactive bond at opposite ends of the molecule. The most significant difference between ATIII and alpha 1-antitrypsin lies in the 45 residue N-terminal extension in ATIII which contribute to the definition of the heparin binding site. This loop region at the surface of the molecule is held by two disulphide bridges to the protein core and exhibits high temperature factor values. It forms a valley which restrains the possibilities for binding of heparin. Docking of the pentasaccharide unit which represents the minimum fragment of heparin able to bind to ATIII indicates a possible role for arginine 14 in the interaction of heparin and the protein.