Intracellular cavities characterized by the presence of microvilli have been identified in dispersed thyroid cells. These structures resembling follicular lumina were called intracellular lumina or ICL. Freshly dispersed cells did not contain ICL. At 37 degrees C, ICL formation was a rapid process. After 60 min of incubation, ICL were present in 15 to 20% of the cells; the number of ICL remained rather constant during 3 to 4 h of incubation. In the presence of thyrotropin, the number of ICL increased with time to reach a value ranging from 40 to 60 ICL per 100 cells after 4 h of incubation. ICL formation was also increased in the presence of dibutyryl cyclic AMP (2 mM). Vinblastine (30 microM), a microtubule-disrupting agent and monensin (30 microM), an ionophore inhibiting Golgi functions blocked the formation of ICL in control and thyrotropin-stimulated cells. Cycloheximide (0.5 mM) and puromycin (0.5 mM) did not inhibit ICL formation in either control or thyrotropin stimulated cells. The iodination capacity of ICL was studied by quantitative electron microscopic autoradiography after incubation of thyroid cells with 125 I-iodide for 2 to 60 min. Radioiodinated products appeared first in ICL. After 1 h of labeling autoradiographic grains were found mainly in ICL (60-70%) and over the cytoplasm. The labeling of ICL was heterogeneous; ICL contained either few or numerous overlapping grains. Whatever the labeling time, a high proportion of ICL (70-80%) were labeled. The labeling of ICL as well as the labeling over the cytoplasm was increased in the presence of thyrotropin and almost completely inhibited in the presence of an iodide trapping inhibitor: sodium perchlorate. Pulse-chase experiments revealed that thyrotropin stimulated the discharge of 125 I-labeled material from ICL.