IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.