Abstract. Mice were infused continuously with graded doses of bovine TSH (bTSH) and changes in plasma concentrations of bTSH and T4 were measured. Then mice infused with 100 mU TSH per day were sacrificed on days 0, 1, 3 and 5 and their thyroids were excised to determine in vitro secretion of T4, T3 and rT3 during 3 h of incubation. At the end of the incubation, thyroidal contents of T4, T3 and rT3 were also determined after pronase digestion. Plasma bTSH levels were increased on day 1 to a level of 110 μU/ml and remained unchanged thereafter. Plasma T4 concentrations increased approximately 2-fold on day 1, but decreased to initial levels on days 3 and 5. Changes in T4 secretion in vitro paralleled those in plasma T4 concentrations; T4 secretion increased 2-fold on day 1, and decreased to the pre-TSH levels on days 3 and 5. In contrast, T3 secretion increased throughout the experimental period. The T3/T4 ratio in thyroidal secretion in vitro was the same as that in thyroidal contents on days 0 and 1 of TSH infusion, but the former was significantly greater than the latter on days 3 and 5. PTU (5.9 × 10−5 m), a known inhibitor of T4 deiodination, added to the incubation media did not affect T4 secretion on days 0 and 1, but increased T4 secretion on days 3 and 5 to the level of day 1, but did not affect T3 secretion. In the presence of PTU, the T3/T4 ratio in thyroidal secretion did not differ from that in thyroidal contents throughout the experimental period. Secretion of rT3 was increased on days 3 and 5 and PTU augmented this increase. Thyroidal content of rT3 was much increased on days 3 and 5. In contrast, methimazole (10−3 m), which does not inhibit in vitro T4 deiodination, added to the incubation media did not affect T4, T3 and rT3 secretion in vitro. When [125I]T4 was added to the media and incubated with mouse thyroid, no labelled products of T4-deiodination were observed to appear in the media even in mice infused with TSH for 5 days. These results suggest that intrathyroidal metabolism of T4 has physiological significance in controlling thyroid hormone secretion at least in TSH-stimulated thyroids.