Rat thyroid follicles were isolated by collagenase digestion and cultured in suspension on agarose for 1-12 days with 0-0.1-1 mU/ml thyrotropin (TSH). After a 4 h exposure to Na125I they were processed for light and electron microscopy, autoradiography and biochemical analysis. Follicular 125I accumulation (A) and organification (PBI) were measured. Thyroglobulin (Tg) content of follicles and 125I-labelled amino acids in Tg were analyzed by high-performance liquid chromatography (HPLC). Without TSH, follicular lumina and cell polarity persisted. From day 3, the rough endoplasmic reticulum (RER) and ribosomes disappeared while autophagic vacuoles appeared: 125I accumulation and PBI were significantly reduced. From day 6, ultrastructural cell dedifferentiation occurred. At day 12, autoradiographic labelling was found over very few lumina; half of the 125I accumulated was still organified. With 1 mU TSH, follicles formed aggregates with narrow densely labelled lumina lined by tall cells. The RER was well developed up to day 12. 125I accumulation, PBI and iodothyronine (T3, T4) formation in Tg remained significantly higher than in follicles cultured without TSH, showing a transient decrease at days 6 and 9. Monoiodotyrosine/diiodotyrosine (MIT/DIT) and T3/T4 ratios in Tg were not modified, suggesting the persistence in the follicles of a significant iodine pool available for iodination. With 0.1 mU TSH, alterations of cell morphology and reduction of functional properties occurred later than without TSH. In the presence of TSH, morphological signs of new follicle formation were seen. These data demonstrate that closed follicles keep their follicular structure up to 12 days of culture, even without TSH. However, TSH is necessary to maintain iodine accumulation and organification.