Background: Thyroid hormone synthesis requires the normal function of thyroid follicular cells and adequate nutritional intake of iodine. For in vitro studies on thyroid cell pathophysiology, the immortalized FRTL5 rat thyroid cell line and a derivative thereof, the PCCL3 cell line, are widely used. However, a permanent human thyroid cell line is currently lacking. A recent report described a cell line obtained from human thyroid cells designated as Cl-huThyrEC. Methods: Four clones of Cl-huThyrEC cells were obtained and cultured in the presence of thyroid stimulating hormone (TSH). The expression of key genes defining the thyroid follicular cell phenotype was determined by reverse-transcription PCR (RT-PCR) in FRTL5, PCCL3, and Cl-huThyrEC cells. The latter were cultured as monolayers and as organoids in Matrigel. Iodide uptake was measured and compared among the cell lines. Results: Gene expression analysis reveals that Cl-huThyrEC cells express the thyroid-restricted transcription factors (PAX8, NKX2.1, FOXE1), the TSH receptor (TSHR), and thyroglobulin (TG), but they do not express the sodium-iodide symporter (NIS), thyroid peroxidase (TPO), and pendrin (SLC26A4). In functional studies, Cl-huThyrEC cells are unable to concentrate iodide. Conclusions: Despite the expression of certain key genes that are limited or restricted to thyroid follicular cells, Cl-huThyrEC cells lack some of the essential characteristics of thyroid follicular cells, in particular, NIS. Hence, their utility as a model system for thyroid follicular cells is limited.