A system of calf thyroid follicular cells in primary cultures has been developed to investigate the control of thyroglobulin gene expression in normal cells in vitro. In low (0.1%) serum conditions, the cells remained quiescent and formed dense aggregates surrounded by slowly spreading cells. High expression of thyroid-specific differentiation markers such as thyroglobulin (Tg) mRNA accumulation and iodide transport required the continuous exposure of cells to thyrotropin (TSH) or other adenylate cyclase activators (cholera toxin and forskolin). In the absence of TSH, Tg mRNA decreased to low but still detectable levels. Addition of TSH, forskolin or cholera toxin restored high Tg gene expression. Hydrocortisone moderately stimulated basal Tg mRNA accumulation and strongly potentiated the effect of TSH. Growth promoters including serum (1-10%), epidermal growth factor (EGF), fibroblast growth factor (FGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) induced calf thyroid cells to develop as a monolayer and inhibited both basal and TSH-stimulated expression of specialized functions. Moreover, only a partial restoration of this expression was achieved after addition of TSH or forskolin to well spread-out cells that had proliferated in response to EGF or serum. The results show that in calf thyroid cells, iodide transport and Tg gene expression are regulated by TSH through cyclic AMP; hydrocortisone potentiates this effect on Tg gene expression, while all growth promoting factors inhibit the expression of these differentiated functions.