In the present study we have obtained several lines of evidence indicating the presence of type II iodothyronine deiodinase (DII) in the mouse mammary gland. 5'-deiodinase activity in the mammary gland has an apparent K(m) value of 4.4 nM for T(4) and is inhibited by aurothioglucose but not by propylthiouracil. These characteristics are similar to those of DII in other tissues. We cloned a 1.4-kb cDNA, which contains the entire mouse DII coding region and has high homology with the rat DII cDNA, from the mammary gland and brain. Northern blot analysis showed the presence of 7.9 kb DII mRNA in the mammary gland and brain. The levels of DII activity and mRNA in lactating gland were significantly lower than those in virgin and pregnant glands, suggesting that DII is regulated at the pretranslational level. In addition, we found the low level of DII enzyme activity and transcript in various other mouse tissues.