The Na1/I2 symporter (NIS) is the plasma membrane protein that mediates active iodide uptake into thyroid follicular cells. To under- stand the molecular mechanisms of human NIS (hNIS) gene regu- lation, the 2-kb immediate 59-flanking region of hNIS was charac- terized. The tentative hNIS transcriptional start site was mapped to 2375 nucleotide relative to the ATG site. Various 59-genomic DNA fragments were tested for promoter activity in thyroid and nonthyroid cells. Our results indicate that the DNA regulatory elements in the 2-kb immediate 59-flanking region were not sufficient to confer thy- roid-selective transcription of the hNIS gene. In addition, hNIS min- imal promoter was localized to a region of 144 bp that contains a 90-bp stretch of a highly conserved DNA fragment between hNIS and rat NIS. (J Clin Endocrinol Metab 83: 3247-3251, 1998)