Thyroglobulin from a human goiter, containing four atoms of iodine/molecule (660,000 daltons), was iodinated with Na 125I and KI in vitro to achieve a net addition of either 2 or 7.8 atoms of iodine/molecule. After fractionation by high performance liquid chromatography, iodinated tryptic peptides from S-cyanoethylated 125I-thyroglobulin were purified, sequenced, characterized by [125I]iodoamino acid distribution, and localized within thyroglobulins primary structure based upon the published cDNA sequence, (Malthiery, Y., and Lissitsky, S. (1987) Eur. J. Biochem. 165, 491-498). The addition of 2 atoms of iodine/molecule of thyroglobulin produced iodotyrosyls at five principal sites, with no 125I-hormone formation. The addition of 7.8 atoms iodinated the same sites more heavily, produced iodotyrosyls at 10 additional sites, and formed iodothyronines at 5 sites. After addition of 2 atoms of iodine, tyrosyl 24 and 11% of thyroglobulins 125I, while tyrosyl 2572 had 24%, but with 7.8 added atoms of iodine, tyrosyl 24 had more of the thyroglobulins [125I]iodothyronine (36 versus 26%). Since tyrosyls 149, 866, and 1466 were iodinated early but did not form the inner rings of iodothyronines, they are attractive candidates for donors of outer iodothyronyl rings. The sequences around the iodotyrosyls fall into three consensus groups, as follows: 1) Glu/Asp-Tyr, associated with synthesis of thyroxine (residues 24, 2572, and 1309), or iodotyrosine (residues 2586 and 991); 2) Ser/Thr-Tyr-Ser, associated with synthesis of iodothyronine (residue 2765) and iodotyrosine (1466 and 883); and 3) Glu-X-Tyr, 7 of the remaining 8 iodotyrosyls occur in this sequence, and we found iodine incorporation at each place this sequence appears in the thyroglobulin molecule. Iodine has been found at homologues of most of these sites in thyroglobulins of other species. We conclude that the primary structure of thyroglobulin, and particularly these consensus sequences, have a major role in the formation of thyroid hormones and their iodinated precursors.