Autoimmune lymphocytic thyroiditis (LT) is a common cause of primary hypothyroidism. Autoreactive T lymphocytes are clearly associated with this disorder, but their pathogenic role in spontaneously occurring LT remains to be established. In the present study, thyroglobulin-reactive T lymphocytes were cultured from young, unprimed LT-prone BB/Wor rats before the age of spontaneously-occurring LT. Although similar investigations have been conducted in animal models for experimental autoimmune thyroiditis (EAT), this study is unique because it examines a mammalian model for spontaneous LT. Splenic T lymphocytes were isolated from unprimed 30 to 45-day-old Fisher and LT-prone BB/Wor rats before the onset of LT, then tested for activation by normal (NL), iodine-poor (LI), and iodine-rich (HI) rat thyroglobulin (Tg) in bulk proliferation assay. BB/Wor rat lymphocytes were activated by all three Tg preparations, but Fisher lymphocytes were unresponsive (BB/Wor vs Fisher; p < 0.0001, Student's t-test). There was no difference between BB/Wor rat responses to the three preparations (p > 0.05, ANOVA). Based on these results, we conclude that Tg-responsive T lymphocytes can be cultured from young, unprimed LT-prone BB/Wor rats. Isolating such lymphocytes before the onset of histologically demonstrable LT strengthens the argument that antigen-specific T cells have a pathogenic role in the development of spontaneous autoimmune thyroid disease. The fact that these lymphocytes recognized normal, iodine-rich and iodine poor preparations of rat Tg equally well suggest that unlike the EAT model, spontaneously-occurring Tg-reactive T cells are not influenced by thyroglobulin's iodine content.