Perchlorate blocks thyroidal iodide transport in a dose-dependent manner. The human sodium/iodide symporter (NIS) has a 30-fold higher affinity for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated. To show this, we incubated NIS-expressing FRTL-5 rat thyroid cells in various concentrations of perchlorate, and we used a sensitive ion chromatography, tandem mass spectrometry method to measure perchlorate uptake in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1 to 10 µM. The perchlorate content from cell lysate was analyzed, showing a higher amount of perchlorate in cells that were incubated in medium with higher perchlorate concentration. Thyroid stimulating hormone (TSH) increased perchlorate uptake in a dose-related manner, thus supporting the hypothesis that perchlorate is actively transported into thyroid cells. Incubation with non-radiolabeled iodide led to a dose-related reduction of perchlorate uptake. To determine potential toxicity of perchlorate, the cells were incubated in 1 nM to 100 µM perchlorate and cell proliferation was measured. Even the highest concentration of perchlorate (100 µM) did not inhibit cell proliferation after 72 hr of incubation. In conclusion, perchlorate is actively transported by NIS into thyroid cells and does not inhibit cell proliferation.