The FRTL-5 cell line is a stable thyroid cell line derived from the thyroid gland of the Fischer rat under defined culture conditions, which has been widely adopted as a model system for the study of thyroid cell function and for bioassay. While characterizing by flow cytometry FRTL-5 cells that were supplied to this laboratory by ATCC (American Type Cell Collection), we discovered that the cells (ATCC CRL8305) were not diploid, having approximately twofold the DNA content relative to a diploid control. The increase in DNA content also applied to cells originally supplied by the ATCC (described as passage 14) that when counted in metaphase had a modal chromosomal count of 84, indicating tetraploid status, double the expected 42 of a diploid rat cell. In a private communication, the ATCC confirmed these findings which nevertheless are contrary to previous literature reports where they were reported to be diploid. Tetraploid cells are less sensitive to thyrotropin (TSH) as measured by cyclic adenosine monophosphate (cAMP) production, compared with diploid cells (p = < 0.001). Despite similar 3H-thymidine uptake in 0.2% fetal calf serum, tetraploid cells show increased 3H-thymidine uptake in 5% fetal calf serum in the absence of TSH (p = 0.001). The origin of these chromosomal changes is unclear, but these findings must raise doubts regarding the suitability of the tetraploid FRTL-5 cell line as a model for studies of human or animal thyroid physiology.