ADAMTS-13 was recently identified as a new hemostatic factor, von Willebrand factor (VWF)–cleaving protease. Either congenital or acquired defects of the enzymatic activity lead to thrombotic thrombocytopenic purpura (TTP). ADAMTS-13 specifically cleaves a peptidyl bond between Y1605 and M1606 in the A2 domain of VWF. Here, we determined the minimal region recognized as a specific substrate by ADAMTS-13. A series of partial deletions in the A2 domain flanked with N- and C-terminal tags were expressed in Escherichia coli and affinity-purified. These purified proteins were incubated with human plasma, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Western blot. Judging from mobility shifts, all constructs except one were cleaved at the expected site. Data suggested that a minimal region as a functional substrate consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and that further deletion of the E1660-R1668 region led to the loss of cleavage by ADAMTS-13. VWF73 was not cleaved by plasma from patients with congenital or acquired TTP, but cleaved by plasma from patients with hemolytic uremic syndrome, suggesting that VWF73 is a specific substrate forADAMTS-13. Thus, VWF73 will be a useful seed to develop a new rapid assay to determine ADAMTS-13 activity.