Background: Macro-thyrotropin (TSH) is a high-molecular-weight form of TSH. Most cases of macro-TSH are TSH complexed with immunoglobulin G. This study was undertaken to characterize macro-TSH. Methods: Blood samples taken from patients with subclinical hypothyroidism were screened for the presence of macro-TSH with the polyethylene glycol method and confirmed with gel filtration chromatography. TSH receptor antibody was quantified with an electrochemiluminescence immunoassay. Binding studies were performed using 125 I-human TSH, and the specificity of anti-TSH autoantibodies was tested by displacement experiments using excess amounts of the unlabeled related peptides. Macro-TSH and serum TSH levels were evaluated twice over a one-to four-year interval. Results: Sixteen patients (11 females and 5 males; aged 8-82 years) were diagnosed as having macro-TSH. None of the patients with macro-TSH tested positive for TSH receptor antibody. Judging from the affinity and the binding capacity of anti-TSH autoantibodies, two classes of binding sites were identified. Regarding specificity, there were anti-human TSH-beta autoantibodies that were partially cross-reactive to bovine and/or rat TSH-beta. There were also autoantibodies against human glycoprotein a, a common subunit among human TSH, luteinizing hormone, and follicle stimulating hormone. Macro-TSH persisted in 11/13 patients who could be reevaluated over a one-to four-year interval after the first evaluation. Serum TSH levels returned to normal in the remaining two patients whose macro-TSH disappeared. Conclusions: It is concluded that anti-human TSH autoantibodies are a major cause of macro-TSH and that macro-TSH may persist for a long time.