ADAMTS13 is a member of the A Disintergrin And Metalloprotease with ThromboSpondin type I repeat family, and it cleaves the Tyr1605-Met1606 bond in the von Willebrand factor (VWF) central A2 domain, thereby decreasing platelet adhesion mediated by VWF. Recently a minimal substrate for ADAMTS13 was characterized that consists of GST linked to Asp1596-Arg1668 with a C-terminal 6×His tag (VWF73). Further removal of 9 amino acids that comprise a predicted C-terminal α-helix (VWF64) appeared to eliminate cleavage by plasma ADAMTS13, suggesting a critical role for this helix in substrate recognition. We obtained similar results, but VWF64 was cleaved significantly at long reaction times. For example, plasma ADAMTS13 (0.3 nM, one-tenth of normal plasma) cleaved 50% of 65 nM VWF73 in 2 hours and 50% of 62 nM VWF64 in 24 hours. Similar results were obtained in either 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.1 μM ZnCl2, or 5 mM Tris-HCl, pH 8.0, 10 mM BaCl2. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. Using 3 nM enzyme for 30 min, VWF73 was cleaved ~2-fold faster by ADAMTS13 truncated after the spacer domain (MDTCS) than by ADAMTS13 truncated after the cysteine-rich domain (MDTC). Conversely, VWF64 was cleaved ~3-fold faster by MDTC than by MDTCS. Also, in 30 min MDTCS cleaved 70% of VWF73 and <10% of VWF64, whereas MDTC cleaved 40% of VWF73 and 30% of VWF64. ADAMTS13 truncated after the first TSP1 repeat (MDT) or the disintegrin domain (MD) had markedly reduced activity, but with prolonged incubation (24 h) at increased concentration (30 nM) both enzymes cleaved most of VWF64 and VWF73 at the expected site. No aberrant cleavage products were detected by Western blotting. The metalloproteinase domain alone (M) was inactive. The selective effects of deleting the cysteine-rich or spacer domain suggest that the C-terminal α-helix of the VWF A2 domain is not essential but facilitates substrate recognition by interacting with specific C-terminal domains of ADAMTS13, particularly the cysteine-rich and spacer domains.