Transcriptional activation by thyroid hormone (T3) receptor (T3R) generally requires the binding of its high affinity ligand. However, we reported previously that chicken T3R alpha (cTaR alpha) and human T3R beta 1 (hT3R beta 1) could activate transcription from several promoters containing T3R response elements (TREs) in a hormone-independent fashion when expressed in rat anterior pituitary GH4C1 cells. In this study we show that rat T3R alpha 1 also activates transcription without T3 in GH4C1 cells and that the oncoprotein v-erbA that is derived from cT3R alpha but does bind T3 is not a constitutive activator in these cells. Increased expression of T3R results in transcriptional activation of both native and minimal promoters, and this activation does not appear to require a defined TRE in the promoter. Because hormone-independent activity is not observed in several other cell lines, this activity may involve specific factors present in GH4C1 cells. Three mutants with single amino acid changes in a 20-amino acid region of the ligand-binding domain of cT3R alpha do not mediate hormone-independent activity. This region is highly conserved within the nuclear receptor family and has been implicated in interactions with other proteins, suggesting participation of other transcription or accessory factors in the hormone-independent activity of T3R. Two of these mutants mediate hormone-dependent transcriptional activation similar to wild-type cT3R alpha. All three mutants interact in vitro with retinoid X receptor beta similar to wild-type cT3R alpha. Our findings suggest that hormone-dependent and hormone-independent transactivation proceed by distinct mechanisms.