Fragments of thyroglobulin containing the autoreactive epitopes were prepared by an existing procedure, plus an ion-exchange chromatography step which increased their purity. Before purification, 39% of the fragmental material was bound by rabbit anti-human thyroglobulin whereas only 8% was bound by autoantibody, thus confirming earlier reports of more limited epitopes for human autoantibodies than heterologous antibody. Thyroglobulin autoantibodies in the globulin fractions from six human sera showed between 70 and 100% cross-reaction with one another, indicating that the majority of antibodies are directed against the same epitopes. The data are consistent with an estimated of two distinct major epitopic specificities with occasional sera having antibodies directed against a third site. Chemical modification of tyrosine residues by iodination did not inhibit the binding of thyroglobulin to either human autoantibodies or rabbit anti-human thyroglobulin; thus tyrosine residues do not contribute significantly to the epitopes for either auto- or heteroantibodies.