Iodide transport defect is a disorder affecting the active transport of iodide, an essential step in the synthesis of thyroid hormones. We have identified novel germ-line mutations in the Na+/I- symporter (NIS) gene from three Japanese patients with iodide transport defect. One patient had a compound heterozygous mutation of T354P/G93R (Gly93-->Arg [GGC-->CGC]), and two sibling patients had a homozygous mutation of G543E (Gly543-->Glu [GGA-->GGA]). G93R and G543E, two novel mutations, are located in the 3rd and 12th transmembrane domains of NIS which are encoded by exons 1 and 13, respectively. The NIS mutants carrying these mutations had minimal iodide uptake activity when expressed in COS-7 cells, confirming that the identified mutations are the direct cause of the iodide transport defect in these patients. Genotyping of unaffected family members and functional assays of co-transfected COS-7 cells indicate that expression of one normal NIS allele in the heterozygote (T354P, G93R, or G543E) is sufficient to maintain active iodide uptake activity. Thus, none of these NIS mutants acts as a dominant-negative mutant.