Four peptides (HTg-1, 1–10; HTg-2, 2547–2558; HTg-4, 2592–2603; HTg-6, 2737–2748) which contain hormonogenic acceptor tyrosine (Tyr) residues and two control peptides (HTg-3, 2582–2591; HTg-5, 2687–2694) of human thyroglobulin (Tg) were synthesized, radioiodinated and their binding with serial anti-human Tg antisera which had been raised in two rabbits (TG-1, TG-2) tested. Although increased binding of each of the six peptides was observed, HTg-4 and HTg-2 had higher binding whereas HTg-1 and HTg-6 showed lower binding with the immune gamma globulin from both rabbits. Each of the six peptides was iodinated with inorganic iodine ( 127I) using the chloramine-T method and the inhibitory activity of each peptide on the interaction between 125I-T 4 and anti-Tg antibodies was tested. At the same time, Tg obtained from a normal thyroid tissue (NTg, iodine content 0.38%) and from a Hürthle cell adenoma (CTg, iodine content 0.000%) were also tested for inhibition of 125I-T 4 binding. 125I-T 4 binding with rabbit anti-Tg antisera was displaced not only by NTg and CTg but also by three out of four hormonogenic peptides. Among the three peptides, HTg-2 had the highest inhibitory activity, inhibiting 125I-T 4 binding to the extent of 21.5% (TG-1) and 16.0% (TG-2). Two control peptides (HTg-3, HTg-5) did not inhibit 125 I-T 4 binding with anti-Tg antibodies. Since each of the four hormonogenic peptides which contain iodinated tyrosines but not thyroid hormones bound with anti-Tg antisera and displaced 125I-T 4 binding from anti-Tg antibodies, it is suggested that the anti-T 4 antibodies in both rabbits are antibodies which recognized the antigenic determinant of not only thyroxyl residues but also of their vicinity.