The antigenic nature of 15 anti-human thyroglobulin (hTg) monoclonal antibody (mAb) epitopes was studied by two different approaches. First, we tested two successive protease-digest products of hTg. Only four mAb from the same cluster of reactivity recognized a low-molecular weight peptide, the other mAb only bound native hTg or high-molecular weight digest fractions. Second, these 15 mAb were used to immunoscreen hTg expression libraries. Only the same four mAb revealed immunoreactive clones corresponding to region 1149-1295 on the hTg primary sequence. After subcloning, this antigenic determinant was reduced to a 102-amino acid peptide (hTg region 1149-1250). The two different methodologies were coherent and complementary, and demonstrated that hTg sequence 1149-1250 is the target for this cluster of four mAb. Moreover, anti-hTg autoantibodies which cross-reacted with these mAb bound the 102-amino acid peptide. This epitope was the one most frequently detected by sera from autoimmune thyroid disease. The data confirm the presence of an immunodominant domain in the central part of the hTg molecule and suggest that this mAb epitope may be a powerful probe for the diagnosis of autoimmune thyroid disorders.