Subcutaneous administration of bovine (b) TSH (up to 10 IU) to 8-week-old male guinea pigs was followed by a transient elevation in serum thyroid hormone levels (T4 and T3) and an increase in thyroid weight (∼25–40%), which returned to control levels by 3 days. Total thyroid TSH receptor content was assessed by the binding of receptor-purified [125I]bTSH to 15,000 × g fractions of thyroid homogenate. The TSH receptor content paralleled the increase in thyroid weight, with no detectable change in the TSH-binding capacity per mg tissue. Intraperitoneal minipump infusions of bTSH (1 IU/day) over 6 days produced marked and persistent increases in thyroid hormone levels and thyroid weight (>300%) and a similar increase in TSH receptor content. There was no change in the single site equilibrium association constant for bTSH [1.1 × 109 M–1 ± (SE) 9.6 × 107 M–1] and no alteration in the binding capacity per mg tissue (67.2 ± 6.4 pg/mg). Investigation of the in vitro adenylate cyclase response to bTSH (10 mU/ml) and the production of immunoassayable cAMP showed no difference between thyroid tissue obtained from bTSH-treated animals and that obtained from untreated control animals. These observations demonstrated that TSH exerted a positive regulatory effect on its receptor and, under the in vivo conditions used, failed to induce TSH receptor loss or physiologically important desensitization. Such data may explain how TSH receptor antibodies are able to act as TSH agonists and maintain increased thyroid hormone output in human disease.