The amino acid sequence established for human thyroglobulin (hTG) from its cDNA sequence contains 20 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of hTG in order to determine which sites are actually linked to carbohydrate. In addition, the distribution of oligosaccharide type(s) at these confirmed sites of N-linked glycosylation has been examined. Glycopeptides were purified using gel permeation chromatography followed by several steps of HPLC. The purified tryptic glycopeptides were characterized by gas phase sequencing and carbohydrate analysis and located within the amino acid sequence of thyroglobulin. Each of the recovered glycopeptides contained a consensus sequence for N-linked glycosylation. Of the 20 putative N-linked glycosylation sites in the human thyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the mature protein. Eight of these confirmed glycosylation sites (at positions 57, 465, 510, 729, 797, 1696, 1754, and 2230) appear to be linked to complex-type oligosaccharide units containing fucose and galactose in addition to mannose and glucosamine. Five sites (at positions 1200, 1329, 1993, 2275, and 2562) contain high mannose type units and two sites (at positions 179 and 1345) are linked to oligosaccharide units containing galactose in addition to mannose and glucosamine but no fucose and may be either hybrid or complex structures. In addition, position 928 was found to be degenerate in oligosaccharide structure and very different oligosaccharide composition types were found associated with peptides containing the same amino acid sequence. A high probability of a beta turn which would include the glycosylated asparagine residue was predicted for the amino acid sequence found at 13 of the 16 sites. The glycosylation pattern in hTG was also compared with the data recently reported for bovine thyroglobulin (bTG) (27) and as has been recently reported for bTG, no oligosaccharides of the high mannose type were found in the N-terminal portion of hTG. Only four of the 20 putative sites the sequence of hTG, at asparagine residues 91, 477, 1849, and 2102 were not represented in the purified glycopeptide population and are presumed to escape significant glycosylation.