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 (5'D2) activity and mRNA are present in the mouse mammary gland and that their levels are reduced in the lactating gland. To investigate the regulatory mechanism of mouse 5'D2 gene (mdio2) expression in mammary epithelium, we employed the 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), prolactin, and epidermal growth factor (EGF), and 5'D2 activity and the D2-to-GAPDH mRNA ratio were measured by ¹²⁵I release from ¹²⁵I-labeled thyroxine and semiquantitative RT-PCR, respectively. EGF increased both 5'D2 activity and mRNA levels about twofold. GC reduced both 5'D2 activity and mRNA in a dose-dependent manner, and their levels were decreased to approximately one-tenth and one-fifth, respectively, of control levels. These data demonstrated that mdio2 expression in HC11 cells is upregulated by EGF mainly at the pretranslational level and downregulated by GC at both pre- and posttranslational levels. Furthermore, we showed that GC reduced the promoter activity of the 627- bp 5'-upstream region of the mdio2/luciferase chimeric reporter gene, suggesting that GC exerts its effect, at least in part, at the transcriptional level.