To investigate the mechanism of I⁻ transport stimulation by TSH, we studied the effects of TSH on Na⁺/I⁻ symporter (NIS) messenger RNA (mRNA) and protein levels in FRTL-5 cells and correlated these with I⁻ transport activity. When 1 mU/ml TSH was added to quiescent FRTL-5 cells, a 12-h latency was observed before the onset of increased I⁻ transport activity, which reached a maximum [∼27 times basal (5H medium) levels] at 72 h. In contrast, Northern blot analysis, using rat NIS complementary DNA as a probe, revealed that addition of TSH to these cells significantly increased NIS mRNA at 3–6 h, reaching a maximum after 24 h (∼5.9 times basal levels). Forskolin and (Bu)₂cAMP mimicked this stimulatory effect on both the I⁻ transport activity and mRNA levels. d-ribofranosylbenzimidazole, a transcription inhibitor, almost completely blocked TSH-induced stimulation of I⁻ transport and NIS mRNA levels. Western blot analysis demonstrated that TSH increased NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of TSH-induced I⁻ transport activity. However, it also showed that the amount of NIS protein already present in FRTL-5 cell membranes before the addition of TSH was about one third of the maximum level induced by TSH. These results indicate that stimulation of I⁻ transport activity by TSH in thyrocytes is partly due to a rapid increase in NIS gene expression, followed by a relatively slow NIS protein synthesis. However, the existence of an abundant amount of protein in quiescent FRTL-5 cells with very low I⁻ transport activity also suggests that this activity is controlled by another TSH-regulated factor(s).