The Na(+)/I(-) symporter (NIS) is a key plasma membrane glycoprotein that mediates active I(-) transport in the thyroid gland (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460), the first step in thyroid hormone biogenesis. Whereas relatively little is known about the mechanisms by which thyrotropin (TSH), the main hormonal regulator of thyroid function, regulates NIS activity, post-transcriptional events have been suggested to play a role (Kaminsky, S. M., Levy, O., Salvador, C., Dai, G., and Carrasco, N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3789-3793). Here we show that TSH induces de novo NIS biosynthesis and modulates the long NIS half-life ( approximately 5 days). In addition, we demonstrate that TSH is required for NIS targeting to or retention in the plasma membrane. We further show that NIS is a phosphoprotein and that TSH modulates its phosphorylation pattern. These results provide strong evidence of the major role played by post-transcriptional events in the regulation of NIS by TSH. Beyond their inherent interest, it is also of medical significance that these TSH-dependent regulatory mechanisms may be altered in the large proportion of thyroid cancers in which NIS is predominantly expressed in intracellular compartments, instead of being properly targeted to the plasma membrane.