The type 2 iodothyronine deiodinase (D2) is critical for the intracellular production of 3,5,3-triiodothyronine from thyroxine. The D2 mRNA of higher vertebrates is over 6 kilobases (kb), and no complete cDNA clones have been reported. Using 5-and 3-rapid amplification of cDNA ends and two cDNA libraries, we have cloned the 6094-base pair full-length chicken D2 cDNA. The deduced protein is ϳ31 kDa and contains two inframe UGA codons presumably encoding selenocysteine. One of these is in the highly conserved active catalytic center; the other is near the carboxyl terminus. Unusual features of the cDNA include a selenocysteine insertion sequence element ϳ4.8 kb 3 to the UGA codon in the active center and three short open reading frames in the 5-untranslated region. The K m of D2 is ϳ1.0 nM for thyroxine, and the reaction is insensitive to inhibition by 6-n-propylthiouracil. Chicken D2 is expressed as a single transcript of ϳ6 kb in different brain regions and in the thyroid and lung. Hypothyroidism increases D2 mRNA in the telencephalon. Unlike in mammals, D2 mRNA and activity are expressed in the liver of the chicken, suggesting a role for D2 in the generation of plasma 3,5,3-triiodothyronine in this species.3,5,3Ј-Triiodothyronine (T 3 ), 1 which accounts for the biological effects of the thyroid prohormone thyroxine (T 4 ), is generated by the removal of a single iodine from its phenolic ring. This deiodination process is catalyzed by two different enzymes, types 1 and 2 deiodinase. Type 1 deiodinase (D1) is a high V max , high K m (ϳ10 M for T 4 ) enzyme, which is highly sensitive to inhibition by 6-n-propylthiouracil (PTU), contains selenocysteine in its active center and can also catalyze the removal of iodine from the tyrosyl ring of T 4 or T 3 or their SO 4 derivatives. The presence of a PTU-insensitive 5Ј-deiodinase termed D2 was first suspected from in vivo studies showing that T 3 generation from T 4 in the hypothyroid rat pituitary was not inhibited by PTU (1). In contrast to D1, D2 has an exclusive 5Ј-activity. It has a low V max with high affinity for T 4 and rT 3 with the K m values in the nanomolar range. D2 plays a particularly important role in the thyroid status of the central nervous system, since it produces more than 75% of the nuclear T 3 in the cerebral cortex (2). To date, D2 activity has been reported in the chicken brain, and the low K m and the increased D2 activity during hypothyroidism are similar to those characteristics of the mammalian D2 (3). Limited information regarding the presence of D2 activity in other avian tissues is available, although PTU-insensitive 5Ј-deiodinase activity has been found in the quail liver (4).In the avian species, as in other vertebrates, thyroid hormone is required for many critical steps in development and maturation (5). Triiodothyronine specifically increases at the time of pipping when the animal begins active respiration (6, 7). This is analogous to the marked increase in circulating T 3 in human neonates, which is thought to occur through a combi...