Abstract Dog thyroid cells in primary culture in a low serum, hormone-supplemented medium represent a model system which allows the direct in vitro study of long-term hormonal effects, both on proliferation and differentiation. The cells exhibited various morphological responses to thyrotropin (TSH): rapid induction of cytoplasmic arborization, persistence of a cuboidal epithelial shape, and formation of domes. Moreover, TSH promoted cell proliferation and biochemical expression of differentiation: high levels of iodide transport and, to a lesser extent, iodide binding to protein. All the TSH effects were completely reproduced by specific activators of adenylate cyclase—cholera toxin and the diterpene forskolin—or by dibutyryl cyclic AMP, which indicates that they are mediated by cyclic AMP (cAMP). We showed that epidermal growth factor (EGF) and pituitary fibroblast growth factor (FGF) are potent mitogens for the dog thyroid cells. Moreover, chronic exposure to EGF induced a striking fibroblast-like morphology and inhibited all the studied characteristics of morphological and biochemical differentiation stimulated by TSH. The effects of EGF were reversible after its wash-out. Other mitogenic treatments, FGF or high serum concentrations, did not reproduce the dedifferentiation effects of EGF, suggesting that they are not directly dependent on mitogenic stimulation. As the effects of EGF were obtained in the range of physiological concentrations, the role of this hormone in the regulation of the thyroid gland is discussed.