The intraluminal distribution of newly synthesized (injection of [3H]leucine) and newly iodinated (injection of Na125I) proteins in thyroids of rats given T4 for 2 days was studied with quantitative electron microscopic autoradiography. Three, 4.5, and 6 h after [3H]leucine about 90%, 85%, and 65%, respectively, of the luminal label was confined to the microvillus region. This distribution differed from that of newly iodinated protein; already 2 min after injection only about 30% of the grains was located over the microvillus region. The remaining 70% of the grains located outside the microvillus region formed a gradient towards the center of the lumen. The grain distributions 30 min and 2 h after Na125I were similar to that present after 2 min. The distribution of grains after pulse labeling with Na125I (injected 2 min before propylthiouracil and 2 h before fixation) was also similar to that found in rats injected with Na125I alone, indicating that diffusion of labeled proteins in the lumen was very slow in T4-treated rats. A slow diffusion was also suggested by the presence of an unlabeled peripheral ring in follicle lumens of T4-treated rats injected with Na125I 48 h before fixation. In normal rats given [3H]leucine 3 h before fixation or Na125I 1 h or 48 h before fixation the grains were homogeneously distributed in most follicle lumens. Together our findings indicate that (1) administration of T4 has effects on the diffusion properties of the colloid; (2) iodine is incorporated not only into newly synthesized thyroglobulin recently delivered to the follicle lumen but also into molecules already stored in the lumen; (3) a portion of the iodine incorporated into proteins is bound to molecules which are not in direct contact with thyroperoxidase in the apical plasma membrane.