Autoradiographs of human goiters demonstrate that the speed of diffusion of newly iodinated thyroglobulin (Tgb) molecules through the colloid space may vary widely from one follicle to another. Since the mechanisms which govern the mixing of the colloid are unknown, we investigated the effect of TSH on these processes in rat thyroid glands. Autoradiographs were prepared from thyroids of rats killed 1 h after 125I or 4 h after [3H]leucine injection. In animals treated with T4 for 2 days, 70% of all follicles showed ring labeling of the colloid periphery with both isotopes, indicating slow mixing of newly synthesized and newly iodinated Tgb molecules with preexisting ones. TSH markedly enhanced the mixing process, thereby diminishing the incidence of ring reactions to roughly 10% of all follicles. These results were expected. Unexpected however, was, the nearly total absence of rings in thyroids treated with T4 for 25 days. Semiquantitative autoradiography revealed a higher absolute number of both newly iodinated and newly synthesized Tgb molecules in the core of follicles in chronically suppressed compared to acutely suppressed thyroids. Moreover, after chronic T4 pretreatment, the effect of TSH on diffusion was the opposite of that observed in acutely T4-treated glands, since 0.5 IU TSH injected twice daily between days 21 and 25 caused the reappearance of 125I and [3H]leucine labeled rings in 44% and 33%, respectively, of all follicles. We conclude that acute TSH suppression slows intraluminal diffusion of thyroglobulin molecules and acute TSH injection accelerates the mixing process, whereas, in contrast, chronic TSH suppression improves and acute TSH action on chronically suppressed follicles impairs diffusion. Therefore, the impact of TSH-mediated processes on the hydrodynamic properties of colloid, and thereby on the intraluminal iodination and coupling process, is more complex than hitherto thought.