We localized the site of type-D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). HTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules, on the base of their D-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6 percent. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin-6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxy-terminal region, starting at residue 2513. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as being LTAGXGLRE (residues 2725-2733, X being Ser2729 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5'-triiodothyronine (T3) (171%) and 3,5,3',5'-tetraiodothyronine (T4) (134%), than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, particularly, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Gly2713-Lys2714 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen, and in the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.