Recently it was reported that the peripheral blood and thyroid gland of patients with Hashimoto's thyroiditis contain activated (Ia+ and/or MLR4+) T cells and high levels of 5/9+ ("helper") T lymphocytes. In normal individuals the 5/9 monoclonal antibody recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5/9+ and 5/9- T lymphocytes were isolated from patients with Hashimoto's disease, and the proliferative response induced by human thyroglobulin was investigated. The results show that the total thyroglobulin-induced lymphocyte DNA synthesis is confined to the 5/9+ T-cell fraction. Further subfractionation of 5/9+ into MLR4+ and MLR4- cells clearly indicates that no substantial differences exist in their proliferative capacities. Whether 5/9, MLR4, and Ia antigens, all expressed on the thyroglobulin-responsive T-cell subset, are involved in thyroglobulin-induced cell proliferation, was also analyzed. Although both 5/9 and MLR4 monoclonal antibodies had no effect, complete inhibition of antigen-induced blastogenesis was observed upon addition of monoclonal antibodies (D1/12 and BT2/9) directed to common determinants of Ia antigens. This inhibitory effect was also observed when T or non-T fractions were separately incubated with the monoclonal antibodies before culture. These results indicate that in humans, as in animals, the major histocompatibility complex may play a role in autoimmune thyroiditis. The data show that (a) the thyroglobulin-induced proliferative response is confined to a subset (5/9+) of T lymphocytes and (b) Ia antigens are involved in thyroglobulin-induced lymphocyte DNA synthesis in Hashimoto's disease.