Infiltration of the thyroid gland by lymphocytes is a hall-mark of autoimmune thyroid disease; it is particularly evident in Hashimoto's thyroiditis but is also seen in most patients with Graves’ disease. Infiltrating cells are comprised primarily of T lymphocytes., of which only a minority appears to be activated. Their precise pathogenic role is largely unknown. Since perforin has been a marker for functionally activated cytotoxic T cells in situ we elected to assess the presence of perforin-containing cells in thyroid-infiltrating lymphocytes and establish their phenotype. Cells were isolated from seven subtotal thyroidectomy specimens, five from patients with Graves” disease and two with Hashimoto's thyroiditis. The novel findings were as follows: CD4+ perforin-containing T cells occurred only in Hashimoto's glands, suggesting a class II-restricted component of cytotoxicity; in Graves' disease, and to a lesser extent in Hashimoto's, perforin-expressing cells were primarily T cell receptor αβ+ CD4- CD8- (double negative); double negative perforin-containing cells in peripheral blood of normal individuals were largely γδ+T cells. In Hashimoto's samples, the predominant population of T cells expressing perforin was CD8+. By comparison, in studies of the synovial fluid of knee joints from patients with rheumatoid arthritis only a minor population of the perforin-containing cells was double-negative. The data suggest significant differences in cytotoxic autoimmune mechanisms between the two autoimmune thyroid diseases. Functional characterization of double-negative T cells is necessary to define their role in autoimmunity.