The sodium/iodide symporter (NIS) mediates active iodide uptake into thyroid follicular cells and is important for the diagnosis and radioiodide treatment of thyroid cancers. In order to better investigate the transcriptional regulation of the NIS gene, we cloned the 3.2 kb 5'-flanking region of the mouse NIS (mNIS) gene in this study. The cloned 5'-flanking region of mNIS shares 68% identity with that of rat NIS (rNIS), yet has little similarity to that of human NIS (hNIS). Based on sequence homology to rNIS, the putative mNIS transcriptional start site is mapped to -97 nt relative to the ATG site. The minimal promoter of mNIS is located within 650 bp of the 5'-flanking region as determined by the transient expression analysis of promoter-reporter constructs. The mNIS upstream enhancer (mNUE) was identified based on sequence homology to rNUE. The mNUE is localized to the region between -3042 and -2809 nt relative to the ATG site and shares 94.4% identity with rat NUE (rNUE), while only 67.8% identity with human NUE (hNUE). It contains two Pax-8 binding sites and a Tax/CREB binding site. The mNUE is also demonstrated to confer thyroid-specific and TSH-responsive transcriptional activity. The high degree of homology in the 5'-flanking region between mNIS and rNIS suggests that mNIS and rNIS share similar mechanisms for transcriptional regulation.