We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5′-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3, 5, 3′-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp 2100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the 22 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) β or JEG nuclear extracts (containing RXRα) and bacterially expressed chicken T3 receptor α1 (TRα) can occupy both half-sites although the 3′ half-site is dominant. T3 causes dissociation of TRα from the 5′ half-site but increases binding to the 3′ half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR115, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3′ half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp 2700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.