Twenty-seven hybridomas secreting monoclonal antibodies (mAb) directed against new antigenic clusters on human thyroglobulin (hTg) were obtained by fusion of the mouse myeloma P3-X63-Ag8 653 with spleen cells from BALB/c mice immunized with a mixture of hTg and six anti-hTg mAb with the aim of masking the corresponding antigenic clusters previously reported. Fourteen mAb were selected, produced in ascitic fluid and characterized. All these mAb were of the IgG1 subclass. Five new antigenic clusters on the hTg molecule were defined by the 14 mAb, extending the initial antigenic map of hTg to 11 clusters. These mAb were used in an attempt to probe the interaction between hTg and the autoantibodies from patients with Hashimoto's thyroiditis who do not recognize antigenic cluster II, a cluster whose recognition by anti-hTg autoantibodies is significantly associated with thyroid disorders.