The THRB gene encodes the well-described thyroid hormone (T3) receptor isoforms TR1 and TR2 and two additional variants TR3 and TR3 of unknown physiological significance. TR1, TR2 and TR3 are bone fide T3 receptors that bind DNA and T3 and regulate expression of T3-responsive target genes. TR3 retains T3 binding activity but lacks a DNA binding domain and does not activate target gene transcription. TR3 can be translated from a specific TR3 mRNA or is co-expressed with TR3 from a single transcript that contains an internal TR3 translation start site. In these studies we provide evidence that the TR3/3 locus is present in rat but not in other vertebrates including humans. We compared the activity of TR3 with other TR isoforms and investigated mechanisms of action of TR3 at specific thyroid hormone response elements (TREs) in two cell types. TR3 was the most potent isoform but TR potency was TRE-dependent. TR3 acted as a cell-specific and TRE-dependent modulator of TR3 when co-expressed at low concentrations. At higher concentrations, TR3 was a TRE-selective and cell-specific antagonist of TR1, 1 and 3. Both TR3 and TR3 were expressed in the nucleus in the absence and presence of hormone and their actions were determined by cell type and TRE structure, whilst TR3 actions were also dependent on the TR isoform with which it interacted. Analysis of these complex responses implicates a range of nuclear co-repressors and co-activators as cell-, TR isoform- and TRE-specific modulators of T3 action.