Type 2 deiodinase (D2) is a low K(m) iodothyronine deiodinase that catalyzes the removal of a single iodine from the phenolic ring of T4 or rT3. We sequenced and subcloned the open reading frame from a partial complementary DNA (cDNA) clone (2.1 kilobases) prepared by Genethon (Z44085) from a human infant brain cDNA library. The open reading frame encodes a putative 273-amino acid protein of 31 kDa with greater than 70% similarity to the Rana catesbeiana D2 protein. Transient expression of the cDNA produces a low K(m) (5 nM for T4; 8 nM for rT3) propylthiouracil- and gold thioglucose-resistant 5'-deiodinase in 293-HEK cells. Human D2, like human type 1 (D1) and type 3 (D3) deiodinases, is a selenoenzyme, as evidenced by 1) the presence of two in-frame UGA codons (positions 133 and 266), 2) the synthesis of a 31-kDa 75Selabeled protein in D2 cDNA-transfected cells, and 3) the requirement for a 3'-selenocysteine incorporation sequence element for its translation. Unlike D1 and D3, we were not able to covalently label overexpressed D2 with N-bromoacetyl [125I]T3 or -T4. We found that the human D2 messenger RNA is 7-8 kilobases and is expressed in brain, placenta, and, surprisingly, cardiac and skeletal muscle. Type 2 deiodinase activity was also present in human skeletal muscle. These results indicate that there are unique features of D2 that distinguish it from the two other selenodeiodinases. The expression of D2 in muscle suggests that it could play a role in peripheral, as well as intracellular, T3 production.