The type 2 iodothyronine deiodinase (D2) catalyzes T₄ activation. In humans, unlike rodents, it is widely expressed, and its action probably contributes to both intracellular and plasma T₃ pools. We have isolated the 6.5-kb 5′-flanking region (FR) and the previously uncloned 553 nucleotides (nt) of the 5′-untranslated region (UTR) of hdio2. The 5′-UTR is complex, with three transcription start sites (TSS) (708, 31, and ∼24 nt 5′ to the ATG), an alternatively spliced approximately 300-nt intron in the 5′-UTR, and three short open reading frames 5′ to the initiator ATG. The previously reported approximately 7.5-kb D2 messenger RNA (mRNA) is actually an approximately 7-kb doublet that is present in thyroid, pituitary, cardiac and skeletal muscle, and possibly brain, but with only the longer transcript in placenta. A canonical cAMP response element-binding protein-binding site is present at about 90 bp 5′ to the most 5′-TSS. It accounts for the robust response of the 6.8-kb hdio2 5′-FR to protein kinase A. Forskolin increases D2 mRNA in human thyroid cells, which may explain the high D2 mRNA in Graves’ thyroid and thyroid adenomas. The hdio2 gene structure and Northern blot results suggest that D2 expression is tightly controlled and tissue specific.