To characterize the structural requirements for the conformational flexibility in plasminogen activator inhibitor-1 (PAI-1) we have crystallized human PAI-1, carrying a mutation which stabilizes PAI-1 in its substrate form. Crystallization was performed by the hanging drop diffusion method at pH 8.5 in the presence of 19% (w/v) polyethyleneglycol 4000 as a precipitant. The crystals appear after 3 days at 23 degrees C and belong to the monoclinic space group C2 with cell dimensions of a = 151.8 A, b = 47.5 A, c = 62.7 A, and beta = 113.9 degrees, and one molecule in the asymmetric unit. The X-ray diffraction data set contains data with a limiting resolution of 2.5 A. Biochemical analysis of the redissolved crystals indicated that during the crystallization process, cleavage had occurred in the active site loop at the P1-P1' position. The availability of good-quality crystals of the cleaved form of this serpin will allow its three-dimensional structure to be solved and will provide detailed information on the structure-function relationship in PAI-1.