The TSH receptor (TSH-R) is the target antigen for disease-related autoantibodies in Graves' disease and primary myxoedema, but the repertoire of the antibodies or the nature of the precise antigenic epitopes is not known. We have immortalized peripheral blood B cells from six different autoimmune thyroid disease patients with Epstein-Barr virus and selected IgG-producing B cells by magnetic selection on anti-IgG-coated beads. Purified recombinant insect cell-derived extracellular region of TSH-R was used to identify the positive wells for expansion in culture. Stable B cell lines (n = 11) were obtained, which after limiting dilution led to two stable B cell clones. B cell lines and clones secreted IgG antibody that were shown to react biochemically with metabolically labeled or in vitro translated, nascent extracellular region of TSH-R, giving strong, confirmatory evidence of the presence of anti-TSH-R antibody. Supernatants from lines contained thyroid-stimulating activity, thyroid-blocking activity (as assessed by inhibition of TSH-mediated cAMP stimulation), or both of these activities. Interestingly, antibodies with stimulating activity were generated from a primary myxoedema patient, and antibodies of blocking specificities were obtained from newly diagnosed thyrotoxic Graves' disease patients. Our results favor a fine balance between stimulating and blocking autoantibody activities in determining the clinical presentation observed in patients with autoimmune thyroid disease patients who have these antibodies present in their serum.