The major targets recognized by anti-centromere autoantibodies are the three centromere-associated proteins (CENPs) A, B, and C, with apparent molecular masses of 19, 80, and 140 kDa, respectively. Previously a major epitope region on the 19-kDa CENP-A antigen was identified by synthesis of a soluble synthetic 15-mer peptide (amino acids 3-17) to be used in enzyme-linked immunosorbent assay and western blot competition assays. However, no systematic experimental scanning for epitope regions on the CENP-A autoantigen has yet been performed. In this study we scanned the complete CENP-A amino acid sequence for epitopes using 19 previously characterized autoimmune-sera. Overlapping peptides 15 amino acids in length and offset by three amino acids were synthesized on activated membranes, covering the whole CENP-A autoantigen. Probing of the membranes with various anti-centromere sera showed that all epitopes are clustered in the N-terminal 45 amino acids. For fine-mapping of this autoreactive region the N-terminus of CENP-A (amino acids 1-45) was scanned again by probing overlapping 15-mer, 12-mer, 10-mer, 8-mer, 7-mer, 6-mer, and 5-mer peptides, all offset by one amino acid, with anti-centromere sera. In this way we localized two epitope core regions within the N-terminal 45 amino acids, one covering amino acids 2-17, recognized by 17 sera, and the other covering amino acids 22-38, recognized by 18 sera. One serum did not react with CENP-A at all. Several sera seem to recognize overlapping individual epitopes within these two epitope core regions. All sera, however, recognize a sequence motif G/A-P-R/S-R-R.