Human thyroglobulin (TG) is one of a growing number of glycoproteins that are known to contain sulfate. Among these TG is unusual because it contains sulfate on both its asparagine-linked complex carbohydrate units and its single chondroitin 6-SO4 unit. We incubated tissue fragments prepared from normal and neoplastic thyroid tissue with [35S]sulfate to study the incorporation of sulfate into these two types of carbohydrate acceptor sites. Incubation conditions (0.1 mM sulfate for 16 h) were selected that maintained linear incorporation of [35S]sulfate and retention of more than 90% of iodinated TG in the tissue. Enzyme susceptibility was used to determine incorporation into the complex carbohydrate units (endoglycosidase-F) and the chondroitin 6-SO4 unit (chondroitin ABC lyase). In a representative experiment, 29.8% of the incorporated sulfate was found in the chondroitin 6-SO4 unit during the first hour of incubation. This increased progressively to 72.5% in the chondroitin unit during the incubation period from 8-16 h. A reciprocal decrease occurred in the proportion of sulfate incorporated into the complex carbohydrate units. TG released into the medium and retained in the tissue had the same ratio of sulfate in the two types of carbohydrate units. Neoplastic thyroid tissue incorporated more [35S]sulfate into TG than normal thyroid tissue from the same patient. Neoplastic and normal tissue differed further in the ratios of sulfate incorporated into the two types of carbohydrate units. We conclude that the incorporation of sulfate into the two types of sulfate-containing carbohydrate units of TG does not occur in a fixed ratio and that this differential incorporation of sulfate does not appear to be related to its release from the tissue.