Abstract Inhibitory antibodies against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura. Epitope-mapping studies revealed that antibodies binding to the cysteine-rich/spacer domains of ADAMTS13 are present in plasma of all patients analyzed so far. We have previously shown that a major antigenic determinant located within the spacer domain. To confirm these results we constructed a recombinant fragment consisting of the disintegrin/TSR1/cysteine-rich domains of ADAMTS13 and the spacer domain of ADAMTS1. The resulting ADAMTS13/ADAMTS1 chimera did not react with IgG present in plasma of a panel of patients with acquired TTP. To identify the amino acid residues that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of 15 hybrids (designated A–O) in which 5–10 amino acids of the ADAMTS13 spacer were exchanged for the homologous sequence of ADAMTS1. Plasma from 6 patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of residues 572–579 (hybrid C) and 657–666 (hybrid M) of ADAMTS13 for the corresponding sequence of ADAMTS1 completely abolished the binding of antibodies from all 6 patients. Other regions of the ADAMTS13 spacer were also involved in binding of antibodies from patient plasma. Regions 580–587 (D), 602–620 (G,H), 629–638 (J), and 667–767 (N) of the ADAMTS13 spacer domain contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). IgG derived from patient 1 required region 602–620 (G,H) for binding to the ADAMTS13 spacer (CGHM-epitope). For antibodies of patient 3, residues 564–571 (B), 580–587 (D), and 629–638 (J) were required (BCDJM-epitope), whereas replacement of residues 602–610 (G) and 629–638 (J) greatly diminished binding of antibodies derived from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of the patients, our results suggest that residues 572–579 (C) and 657–666 (M) comprise a common antigenic core region within the spacer domain that is crucial for binding of anti-ADAMTS13 antibodies in all six patients. Amino acid residues derived from other regions that spatially surround this common antigenic core further modulate binding of antibodies to the spacer domain.