The in vitro and in vivo detection of visible photons from radioisotopes using optical techniques is a fast-growing field in molecular imaging. Tc99m-pertechnetate is used as an alternative to I123 in imaging of the thyroid and is generally imaged with gamma cameras or single photon emission tomography instruments. The uptake in the thyroid tissue is mediated by the sodium-iodide symporter (NIS), a glycoprotein that actively mediates iodide transport into the thyroid follicular cells and several extrathyroidal tissues. The luminescence of the gamma emitter Tc99m-pertechnetate in order to visualize its biodistribution in healthy small living animals by using a commercial optical imaging system is investigated. Here we show that in Nu/Nu mice, the uptake of Tc99m-pertechnetate in the thyroid gland and in salivary glands is very detectable by using radionuclide luminescence imaging. We also found light emission from the stomach in accordance with the literature. The localization of the light signals in the anatomical regions where the radiopharmaceutical is expected, confirmed by resections, shows that it is possible to image NIS-expressing tissues.