Thyroid hormone receptors (TRs) regulate gene expression by binding to specific DNA sequences, denoted thyroid hormone response elements (TREs). The accepted paradigm for TRs proposes that they bind as homo- or hetero- dimers to TREs comprised of two AGGTCA "half-site" sequences. In the prototypic TRE, these half-sites are arranged as direct repeats separated by a 4-base spacer. This dimeric model of TR binding, derived from analysis of artificial DNA sequences, fails to explain why many natural TREs contain more than two half-sites. Therefore, we investigated the ability of different TR isoforms to bind to TREs possessing 3 or more half-sites. We report that the TR isoforms (TR0, TR1, TR2), but not TR1, can bind to reiterated DNA elements, such as the rat GH-TRE, as complexes trimeric or greater in size. The TR0 isoform, in particular, formed homo- and hetero-trimers (with the retinoid X receptor) with high efficiency and cooperativity, and TR0 preferentially used reporters containing these reiterated elements to drive gene expression in vivo. Our data demonstrate that TR isoforms can form multimeric receptor complexes on appropriately reiterated DNA response elements, providing a functional distinction between the TR isoforms and an explanation for TREs possessing three or more half-sites.