Patients with Graves' disease and exophthalmos demonstrate delayed hypersensitivity to antigens present in extracts of certain normal human tissue; namely,thyroid gland and retroorbital tissue. The delayed hypersensitivity can be assayed in vitro by quantitating the amount of a lymphokine, migration inhibition factor (MIF), which is produced when T lymphocytes of patients with Graves' disease and exophthalmos are exposed to these antigens. In the present report, a partial purification is described for the retro-orbital tissue antigen which is responsible for the positive leucocyte migration inhibition factor assay (MIF assay) exhibited by a sensitized lymphocytes of these patients. The purified retro-orbital tissue antigen preparation demonstrates a 50- to 150-fold higher specific activity over crude homogenates in its ability to act as an antigen in the MIF assay of exophthalmic patients. Immunodiffusion, ultracentrifugation, and disc electrophoretic data indicate that this purified antigen preparation, obtained from normal human, retro-orbital tissue, contains thyroglobulin or a derivative of thyroglobulin; immunofluorescence studies localize the anti-thyroglobulin reactive material to the plasma membranes of extraocular muscle fibers of normal individuals. On the basis of these data it is concluded that thyroglobulin or a derivative of the thyroglobulin molecule is present in the orbital muscle of normal individuals. Since thyroglobulin purified from normal human thyroid glands and the purified retro-orbital tissue preparation are nearly equivalent as antigens in the MIF assay of exophthalmic patients, we conclude that thyroglobulin or an antigenic component of the thyroglobulin molecule is one of the antigens to which patients with Graves' disease and exophthalmos demonstrate delayed hypersensitivity.