Human thyroglubulin labelled in vivo by 125I was purified from eight different thyroid glands including normal thyroid tissue, thyrotoxic goitre and euthyroid multinodular goitre. The purified protein was cleaved with cyanogen bromide (CNBr) and the resulting peptides were separated by column chromatography and ion exchange chromatography. Reproducible elution profiles of both protein and iodine were obtained. However, the distribution of iodine depended on the iodine content of the intact thyroglobulin. Small CNBr peptides seemed to be preferentially iodinated, but with a limited capacity. With higher degrees of iodination, larger peptides became richer in iodine. This suggests sequential iodination of the thyroglobulin molecule. The mixture of small peptides was digested by trypsin. Two iodopeptides were identified in this material by peptide mapping and they had identical migration in thyroglobulins of different origin. One of them was purified by ion exchange chromatography and high voltage electrophoresis. Analogous amino acid composition was obtained for the iodopeptide purified from two different thyroglobulins. The data indicates that thyroglobulin iodination occurs in specific portions of the polypeptide chain and probably in a sequential manner.