We prepared 3 samples of 19S thyroglobulin (Tg), 1 from a patient with Graves' disease, another from a patient with nontoxic goiter, and the third from a pool of Tg from normal subjects, and used each Tg preparation to produce a polyvalent antiserum in rabbits. The 3 antisera were similar to each other in their reactivity with thyroid Tg samples from 25 patients with various thyroid disorders and from 10 normal subjects. However, the immunoreactivity of the 35 individual Tg samples varied considerably. Decreased reactivity was associated with proteolysis during Tg preparation, iodination in vitro with 20 or more atoms of iodine/molecule Tg, the 27S species of Tg, Tg from 3 patients with thyroid cancer, and Tg from several patients with Graves' disease. The antiserum to Graves' Tg contained some antibodies that did not bind normal Tg on an affinity column, and these antibodies reacted more with Tg from patients with Graves' disease than with Tg from normal subjects or patients with nontoxic goiters. Thus, Tg from patients with Graves' disease may contain antigenic sites that are not present or exposed in Tgs from other subjects. We conclude that thyroid Tgs from patients with Graves' disease and from those with thyroid cancer may be different in structure from the Tgs of normal subjects. This conclusion is important to an understanding of Tg structure in thyroid disease and to the use of thyroid Tg for preparation of antisera and standards for measuring serum Tg concentrations.