The retinoid X receptor (RXR) isoform RXRgamma has limited tissue expression, including brain, skeletal muscle, and anterior pituitary gland. Within the anterior pituitary gland, RXRgamma expression is limited primarily to the thyrotropes. In this report, we have isolated approximately 3 kb of 5'-flanking DNA of the mouse RXRgamma1 gene. We have identified the major transcription start site in the thyrotrope-derived TtT-97 cells. Transient transfection studies show that a 1.4-kb promoter fragment has full promoter activity in TtT-97 cells. This promoter has much less activity in thyrotrope-derived alphaTSH cells, pituitary-derived GH3 somatomammotropes, and non-pituitary CV-1 cells. None of these cell lines has detectable RXRgamma1 mRNA. A previous report has identified a non-consensus direct repeat (DR-1) element in the RXRgamma2 gene promoter region that mediates stimulation of promoter activity by 9-cis-retinoic acid (9-cis-RA). Inspection of the RXRgamma1 promoter region revealed a non-consensus DR-1 element at -232 bp from the transcription start site. Interestingly, RXRgamma1 promoter activity was suppressed 50% by 9-cis-RA in the TtT-97 thyrotropes. Further experiments in non-pituitary cells showed that suppression of RXRgamma1 promoter activity was RXR-dependent. Mutagenesis of the DR-1 element abrogated suppression of promoter activity by 9-cis-RA, suggesting that this negative regulation requires both RXR and this specific DR-1 element. In summary, we have isolated the mouse RXRgamma1 gene promoter region and identified the major start site in thyrotropes. Promoter activity is uniquely suppressed by 9-cis-RA through a DR-1 element. Isolation and characterization of the mouse RXRgamma1 promoter region provides a tool for further investigation focusing on thyrotrope-specific gene expression as well as negative regulation of genes by retinoic acid.