Technological advances have drastically decreased the number of cells required to analyze expression of the genes and functions of the encoded proteins, making even a small organ like a mouse thyroid amenable to studyin vitro.We have established primary cultures of mouse thyroids that showed, for up to 14 days after seeding, strong cytoplasmic staining for thyroglobulin. The staining then gradually decreased and was present in only 5-10% of thyrocytes at day 28. Furthermore, cultured thyrocytes expressed the thyroperoxidase and thyrotropin-receptor genes, and, although at lower levels, the sodium-iodide symporter gene. Finally, cultured thyrocytes could be transiently transfected by lipofection, using FuGENE 6. Thus, we report that it is possible to cultivate functional primary mouse thyrocytes that can be used for a variety of biological studies. This system is appealing because it permits the use of the ever-increasing number of transgenic, knock-out and knock-in mouse strains in studying thyroid pathophysiology.