Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.