Thyroid hormones are important for mammary gland growth and development. The iodothyronine deiodinases play a key role in thyroid hormone metabolism. We have showed that type II 5'-deiodinase (D2) activity and mRNA are present in the mouse mammary gland and their levels are reduced in the lactating gland. In order to investigate the regulatory mechanism of mouse D2 gene (mdio2) expression in mammary epithelium, we employed HC11 cell line, which is derived from mouse mammary epithelial cells and retains the ability to express differentiated function. HC11 cells were treated with combinations of insulin, glucocorticoid (GC, dexamethasone), PRL and EGF, and 5'D2 activity and D2/GAPDH mRNA ratio were measured by ¹²⁵I^- release from ¹²⁵I labelled T4 and semiquantitative RT-PCR, respectively. EGF increased both D2 activity and mRNA levels about 2-fold. GC reduced both D2 activity and mRNA in a dose dependent manner and their levels were decreased approximately one-tenth and one-fifth, respectively, of the controls. These data demonstrated that mdio2 expression in HC11 cells is up-regulated by EGF mainly at pre-translational level and down-regulated by GC at both pre- and post-translational levels. Furthermore, we showed that GC reduced the promoter activity of 627 bp 5'-upstream region of mdio2 / luciferase chimeric reporter gene, suggesting that GC exerts its effect, at least in part, at the transcriptional level.