Primary cultures of rat thyroid cells were made in medium supplemented with 0.1-0.5% calf serum and containing six hormones or growth factors: insulin, thyrotropin, transferrin, hydrocortisone, somatostatin, and glycyl-L-histidyl-L-lysine acetate. The FRTL strain was purified by successive colonial isolations and was found to maintain highly differentiated features (secretion into the culture medium of physiological amounts of thyroglobulin and concentration of iodide by 100-fold). The

Iodide (I-) uptake by FRTL-5 cells, a functioning rat thyroid cell line, is TSH dependent. The effects of TSH withdrawal are not apparent until 1 day; 1 week is required to reduce I- uptake to a minimal level. The readdition of TSH leads to a return of the I--concentrating ability after a latency of 12-24 h. The reappearance of I- uptake induced by TSH is mimicked by (Bu)2cAMP and agents that elevate intracellular cAMP levels in these cells, such as forskolin, cholera toxin, and a Graves' disease serum. The appearance of I- uptake after TSH occurs 12 h after the appearance of TSH-induced [35S]methionine incorporation. Cycloheximide blocks both the TSH- and (Bu)2cAMP-induced increases in methionine incorporation and I- uptake to the same extent and in an identical concentration-dependent manner. TSH-induced [35S]methionine incorporation is associated with increased radiolabeling of several specific proteins, as revealed by gel electrophoresis; none, however, is radiolabeled coincident in time with the appearance of TSH-induced I- uptake. Several proteins whose apparent synthesis is induced by TSH also exhibit TSH-dependent phosphorylation.