The effect of epidermal growth factor (EGF) on the morphology of porcine thyroid follicles cultured in collagen gel was studied by light and electron microscopy. Treatment with EGF (10 ng/ml) for 1-4 days induced a progressive migration of thyrocytes radiating from the mother follicles into the collagen lattice. Migrating cells were often connected with junctional complexes, thus forming small follicles containing microlumina, in which the limiting portion of the plasma membrane expressed microvilli. Autoradiography of [H-3]thymidine incorporation in EGF-treated cultures showed that more than 80% of nuclei in mother follicles and migrating cells were labeled after 4 days. TSH (2 mU/ml) given simultaneously with EGF did not influence the effect of EGF on multiplication, migration, and formation of microfollicles. TSH given for 12 h after 4 days of exposure to EGF was able to induce luminal dilation of mother follicles, but caused no change in the appearance of microlumina. It is concluded that thyrocytes in collagen gel retain epithelial characteristics during prolonged stimulation with EGF, in spite of a migrating response. The whole sequence of events, starting with the intact follicle, i.e. multiplication, migration of thyrocytes, and formation of new follicles, could, thus, be visualized to occur in response to a single mitogen, EGF. EGF might be involved in the generation of new follicles in the intact gland.