Radioiodine (RAI) treatment has increasingly been used mostly in elderly patients with multinodular goiter (MNG) as an alternative for surgery. Recombinant human thyrotropin (rhTSH) has been demonstrated to increase the uptake of RAI and also to promote a more even distribution of radionuclide among the various nodules. We have compared the surge of autoantibodies to thyroid peroxidase (anti-TPO) and to the TSH receptor (TRAb) in two groups of patients with MNG. Group RAI (n = 15) received only RAI, and Group RAI+rhTSH (n = 15) received RAI 24 h after 0.45 mg of rhTSH intramuscularly. At baseline, all 30 patients had negative anti-TPO antibodies. After RAI, 16 patients (eight in each group) exhibited a positive anti-TPO test (range, 70-2359 U/mL). In the rhTSH-treated group, anti-TPO values were significantly higher (as compared to basal levels; p < 0.02) after 3 months of RAI treatment. After 12 months, the anti-TPO values decreased to lower but still positive concentrations in nine patients (Group RAI: three patients; Group RAI+rhTSH: five patients). Only one patient had a positive TRAb test at baseline (67.5% inhibition of the TSH binding). After RAI, positive TRAb values were present in 21/30 patients. After 6 months of RAI treatment, there was a significant increase of the TRAb values in Group RAI+rhTSH patients. After 12 months, only four patients had positive TRAb (Group RAI: three patients; Group RAI+rhTSH: one patient). Two patients, one of each group, had an elevation of free T4 levels and suppressed serum TSH values, indicating hyperthyroidism (Graves' disease). Bioassay of TSH receptor (TSHR) indicated absence of a significant elevation of cAMP in the medium before and after RAI treatment in all patients. Moreover, predominantly blocking TSHR autoantibodies were detected in six of the 30 patients (three of each group). Sera from these patients were able to reduce the TSH-stimulated cAMP generation by CHO cells. We conclude that the autoantibodies to the TSHR and to TPO may occur after RAI treatment of patients, either with or without previous stimulation by rhTSH. The antibodies to the TSH comprised a combination of agonist (stimulating) and antagonist (blocking) antibodies, which in most patients did not induce clinical and laboratory evidence of active Graves' disease.