Bovine thyrotropin (bTSH) was administered to 17 patients who had thyroid carcinoma. Anti-bTSH antibodies in the patients' sera were detected by three methods: 1) cross-reaction of sera in a homologous bTSH RIA, 2) [125I]bTSH binding to the patients' sera using charcoal to separate bound from free fractions, and 3) gel filtraton to detect [125I]bTSH bound to anti-bTSH in patients' sera. Immunoreactive anti-bTSH antibodies were thus demonstrated in 14 patients. These patients showed specific binding of their sera to [125I]bTSH with the charcoal separation of free from bound hormone. A high titer of antiserum (1:10,000) was found in those patients whose sera reacted strongly in the bTSH RIA (greater than 50 mu U/ml). The binding capacity of the serum of 1 patient was estimated as 2,600 mu U/ml serum. Ten of the patients' sera which showed immunoreactivity to bTSH neutralized bTSH activity in the McKenzie mouse bioassay but did not neutralize the activity of human TSH in this bioassay. Repeated administration of bTSH to 14 patients resulted in development of immunoreactive and neutralizing anti-bTSH antibodies. Development of immunological resistance to bTSH appears inevitable in patients who receive repeated injections of this hormone. Because of the loss of effectiveness of bTSH by antibody formation, the repeated diagnostic and therapeutic use of bTSH is not recommended.