The 22 kDa fragment (Asn₁—Met₁₇₁) purified from iodine-poor human thyroglobulin (hTg) is capable by itself to synthesize thyroxine at Tyr₅, the preferential hormonogenic acceptor site of the protein, after iodination in vitro. To identify the corresponding donor site in this model we studied the fate of the six Tyr residues present in the 22 kDa peptide after in vitro hormone synthesis. Structural studies of the tyrosyl peptides showed that Tyr₅ was the only thyroxine-forming site, the other tyrosines (29, 89, 97 and 107) were noniodinated and Tyr₁₃₀ was recovered in alanine form after CNBH₄ treatment of the Tyr₁₃₀-containing peptide. Taking into account that alanine could arise from aminoreduced pyruvate species, these results showed that in the 22 kDa fragment (1) hormone formation involves the couple Tyr₅ (acceptor)-Tyr₁₃₀ (donor), and (2) dehydroalanine, the resultant product of donor tyrosine after hormone synthesis, has evolved in pyruvoyl form. To test whether Tyr₁₃₀ could also act as donor in hTg hormone synthesis, the 22 kDa peptide was isolated from hTg iodinated under conditions leading to iodotyrosine formation followed or not by hormone formation and the tyrosyl peptides were analyzed. After hTg iodination and before coupling (i.e. hormone synthesis) only Tyr₅ and Tyr₁₃₀ were recovered in iodotyrosine form; after coupling thyroxine was found at Tyr₅ whereas Tyr₁₃₀ disappeared. Taken together these results, correlated with the previously reported cleavage of hTg chain at Tyr₁₃₀ occurring during in vivo hormone synthesis, support the theory that the couple Tyr₅ (acceptor)-Tyr₁₃₀ (donor) would be the preferential hormonogenic site in human Tg.