A 19-residue, thyroxine (T4)-containing peptide, Tryp-T4, has been isolated from the tryptic digest of a low molecular weight, iodine-enriched fragment derived from 19S bovine thyroglobulin. This tryptic peptide represents the only site of significant iodination in the parent polypeptide fragment. The amino acid sequence of the tryptic peptide has been determined and is NH2-Asn-Ile-Phe-Glu-T4-Gln-Val-Asp-Ala-Gln-Pro-Leu-Arg-Pro-Cys-Glu-Leu-Gln- Arg-COOH. The carboxyl-terminal sequence of this peptide shows a high probability of a beta-turn. These findings establish the involvement of at least a single unique sequence within thyroglobulin in thyroxine biosynthesis and the general nature of a hormonogenic site within this protein. This sequence contains at least 30% of the thyroxine present in 19 S bovine thyroglobulin.