There are three known isoforms of the retinoid-X receptor (RXR): RXRα, RXRβ, and RXRγ. RXRα and RXRβ messenger RNAs are widely expressed, whereas RXRγ messenger RNA is restricted to only a few tissues, including embryonic pituitary gland. Little is known about the level of expression and cell distribution of RXR proteins in the adult pituitary gland. To examine these issues further, we raised isoform-specific polyclonal antibodies against each of the known mouse RXR isoforms using synthetic peptides containing isoform-specific epitopes from the amino-terminal region. The specificity of each antibody was confirmed by immunoprecipitation, Western immunoblot analysis, and electrophoretic mobility shift assay with supershift studies of in vitro translated RXR isoforms. Immunocytochemical analysis showed that anti-RXRα and anti-RXRβ antisera stained the nuclei of most pituitary cells. In contrast, anti-RXRγ antiserum stained the nuclei of only a few cells throughout the pituitary. In the hypothyroid state, however, a marked increase in both the number and density of RXRγ-immunostained nuclei were observed compared to those in the euthyroid state. Double immunostaining studies of hypothyroid rat pituitary with antibodies against pituitary hormones indicated that RXRγ protein was predominantly expressed in thyrotropes. Antibody supershift experiments using nuclear extracts of adult rat whole pituitary and rodent pituitary cell lines showed that anti-RXRγ antibody could alter the mobility of protein-DNA complexes formed only from nuclear extracts of rat whole pituitary and thyrotropic TtT-97 cells. In contrast, anti-RXRα and anti-RXRβ antibodies could supershift protein-DNA complexes formed from nuclear extracts of all cell lines tested. RXRγ protein expression in TtT-97 cells also was observed by Western immunoblot analyses. Therefore, there is thyrotrope-predominant expression of RXRγ protein. We speculate that RXRγ may play a role in the regulation of thyroid hormone target genes in thyrotropes and possibly cell type differentiation in the pituitary.