TSHβ is a subunit of TSH that is uniquely expressed and regulated in the thyrotrope cells of the anterior pituitary gland. Thyroid hormone receptors (TR) are known to mediate T₃ suppression of TSHβ gene expression at the level of promoter activity. The role of other nuclear receptors in regulation of this gene is less clearly defined. Retinoid X receptors (RXR) are a family of nuclear transcription factors that function both as 9-cis-retinoic acid (RA) ligand-dependent receptors and heterodimeric partners with TR and other nuclear receptors. Recently, the RXR isoform, RXRγ, has been identified in the anterior pituitary gland and found to be restricted to thyrotrope cells within the pitutiary. In this report, we have further characterized the distribution of RXRγ1, the thyrotrope-restricted isoform of RXRγ, in murine tissues and different cell types. We have found that RXRγ1 mRNA and protein are expressed in the TtT-97 thyrotropic tumor, but not the thyrotrope-variant αTSH cells or somatotrope-derived GH3 cells. Furthermore, we have studied the effects of RXRγ1 on TSHβ promoter activity and hormone regulation in these pituitary-derived cell types. Both T3 and 9-cis-RA independently suppressed promoter activity in the TtT-97 thyrotropes. Interestingly, the combination of ligands suppressed promoter activity more than either alone, indicating that these hormones may act cooperatively to regulate TSHβ gene expression in thyrotropes. The RXRγ1 isoform was necessary for the 9-cis-RA-mediated suppression of TSHβ promoter activity in αTSH and GH3 cells, both of which lack this isoform. RXRβ, a more widely distributed isoform, did not mediate these effects. Finally, we showed that the murine TSHβ promoter region between −200 and −149 mediated a majority of the 9-cis-RA suppression of promoter activity in thyrotropes. This region is distinct from the T₃-mediated response region near the transcription start site. These data suggest that retinoids can mediate TSHβ gene regulation in thyrotropes and the thyrotrope-restricted isoform, RXRγ1, is required for this effect.