Expression of the sodium iodide symporter (NIS) gene in tumor cells may provide a novel mechanism for treating cancer. The NIS mediates the normal physiological transport of iodide across the thyroid cell membrane. This mechanism of iodide uptake has been used to both diagnose and treat thyroid cancer. Tissue expression of the NIS is largely limited to the thyroid; therefore, expression of the NIS gene in cancer cells would allow for specific iodine uptake, radioisotope accumulation, and treatment. In this study, we directly compared the human and rat NIS (rNIS) for their ability to concentrate radioisotope into human and rodent cancer cells. Perchlorate-sensitive (125)I uptake in multiple cell lines was demonstrated following transduction with retroviral vectors expressing either the human or rNIS gene. Surprisingly, iodine uptake was consistently higher with the rNIS gene, up to 5-fold greater, when compared to the human gene, even within a variety of human tumor cell lines. This iodine uptake allowed for cell killing following (131)I treatment in NIS-transduced cells when assayed by in vitro clonogenic assays. These results demonstrate that the rNIS gene provides superior iodine uptake ability, and may be preferable for use in designing anticancer gene therapy approaches.