INTRODUCTION: Thyroid hormone (T3) plays a critical role in gut development and homeostasis. T3 action is mostly mediated through its nuclear receptors, TRα1 and TRβ1, the bona-fide TR isoforms that contain both DNA and hormone binding domains. However, it has recently become clear that several TR variants also exist, notably TRΔα1 and TRα2, which lack the DNA and/or hormone binding domains. The present studies were undertaken to define the role of these TR variants in the context of a T3-regulated gut-specific gene, intestinal alkaline phosphate (IAP), an enterocyte differentiation marker that limits fat absorption. METHODS: Transient transfections were performed in Caco-2 cells with an IAP-Luciferase reporter plasmid and the bona-fide TRα1 +/- the TR variants. RNA was extracted and subjected to RT-PCR to assess effects on endogenous gene expression. EMSA were performed with radiolabeled DNA corresponding to the IAP T3 response element (IAP-TRE) and in vitro synthesized proteins. RESULTS: IAP-Luc reporter assays showed that TRα1 activated the IAP gene approximately 8-fold, confirming our previously published data. RT-PCR (standard and real time) also confirmed the endogenous IAP gene induction by TRα1. The TR variants, TRΔα1 and TRα2, had no effects alone on IAP activation, but in co-transfections each was able to inhibit the TR1-mediated activation of IAP. These effects of the TR inhibitors were directly related to the dosage of plasmid transfected. In addition, real-time PCR demonstrated that the TR variants caused dramatic inhibition of TRα1-mediated endogenous IAP gene activation (approximately 80% and 60%, respectively). This inhibition of IAP gene activation was specific to the T3 pathway, since there was no inhibition of the Cdx1 transcription factor-mediated IAP activation. As expected, EMSA confirmed that TRα2, but not TRΔα1, binds to the IAP-TRE. CONCLUSION: The two naturally occurring TR variant isoforms (TRΔα1 and TRα2) repress TRα1-mediated activation of the IAP gene through distinct mechanisms, indicating that there is a complex interplay among the various TR proteins in modulating the physiological effects of T3 in the gut.