Thyroid follicle cells from various mammalian species incorporate 35-SO4(2-). Light and electron microscopic autoradiographs show that the Golgi complex is the predominant site of sulfate incorporation and that the secretory product accumulating in the follicle lumen is sulfated. In order to determine which components of the luminal content carry the sulfate residues, inside-out follicles from pig thyroid glands were incubated in the presence of 35-SO4(2-) and the secretory product released into the culture medium was analyzed by polyacrylamide gel electrophoresis. The observations show that the secretory product consists of sulfated thyroglobulin and that approximately 13 sulfate residues are bound covalently to 1 molecule of dimeric thyroglobulin. Digestion of 35-SO4(2-)-thyroglobulin with endoglycosidase H removes 20 to 30% of the radioactivity, indicating that the high mannose carbohydrate side chains carry sulfate residues. The complex carbohydrate side chains are apparently free of sulfate since treatment with endoglycosidase D did not alter the sulfate content. About 2/3 of the sulfate is cleaved by hydrolysis with 1 M HCl (5 min, 95 degrees C) indicating the presence of tyrosine sulfate. Part of the sulfate is exposed and presumably located on the surface of the thyroglobulin molecule as suggested by the direct accessibility of 35-SO4(2-)-thyroglobulin to digestion with sulfatases. The sulfate residues contribute to the anionic state of thyroglobulin. It is postulated that the sulfate residues operate in the regulation of thyroglobulin transport in the cell and in the tight packaging of thyroglobulin in the follicle lumen.