The selenoenzyme thyroxine 5'-deiodinase type I deiodinates the prohormone thyroxine to the active thyroid hormone 3,3',5-triiodothyronine. It is thus one of the key enzymes involved in the triiodothyronine-mediated control of growth, differentiation and basal metabolism in vertebrates. We report here the identification of the transcription start site and the cloning of 1500 bases of the upstream regulatory region of the human 5'-deiodinase gene. They contain a complex triiodothyronine-responsive element at nucleotides -696 to -673, consisting of an ideal direct repeat (DR) of two AGGTCA half-sites with a spacing of four nucleotides (DR+4) and a third putative AGTTCA half-site with a spacing of another two nucleotides (DR+2). The whole DR+4+2 specifically bound to thyroid hormone receptor and retinoid X receptor in electrophoretic mobility shift assays. The DR+4+2 mediates triiodothyronine-responsiveness in cotransfection experiments of constructs containing the 5'-deiodinase upstream promoter and enhancer region fused to luciferase or chloramphenicol acetyltransferase reporter genes with expression plasmids of thyroid hormone receptor subtypes. Also, an about 2.5-fold induction of the 5'-deiodinase-promoter-luciferase-reporter construct by all-trans retinoic acid was observed in a cotransfection assay with retinoic acid receptors. Point mutation analysis of the DR+4+2 type hormone-responsive element, however, revealed that it does not alone mediate the retinoic acid effect. The transcription start point of the 5'-deiodinase gene was mapped to nucleotides -23 and -24. No CAAT or TATA box is located within the usual distance to the transcription initiation site. Two GC boxes were found at nucleotides -68 to -63 and -39 to -34. Transfection analysis revealed that the proximal 105 nucleotides in the 5'-flanking region of the 5'-deiodinase gene act as a functional core promoter. This data indicates that triiodothyronine, the end product of thyroid hormone synthesis, positively regulates one of the key enzymes in its production.