The human glycoprotein hormone alpha-subunit (GSU) gene is transcriptionally regulated by glucocorticoids in a cell-type specific fashion. In direct contrast to repression of GSU by glucocorticoids in placenta, GR-modulation in the pituitary is little understood. We show that glucocorticoids stimulate the GSU promoter in immortalized pituitary gonadotrope-derived LT2 cells, while estrogens, androgens and progestins have no significant effect. Moreover, glucocorticoid receptor (GR) acts in a dose-dependent manner at physiological concentrations of glucocorticoids. Transient transfection of GR with Dex treatment further stimulates the GSU promoter, but this induction is severely diminished using a receptor mutated in the DNA-binding domain. Truncation and cis mutations demonstrate that glucocorticoid response element 2 (GRE2) and cAMP-response element 2 (CRE2) within -168 bp of the human GSU promoter are critical for induction. Moreover, dominant-negative CREB markedly inhibits basal, but also Dex induction, of GSU promoter activity. Additionally, GR specifically binds to GRE2 in the human GSU promoter in vitro, and to the 5' region of the endogenous mouse GSU gene, in vivo. Furthermore, overexpression of the homeobox factor, Distal-less 3 that regulates this gene in placental cells through a site partially overlapping GRE2, blocks Dex induction of GSU in gonadotrope cells, indicating that placenta-specific expression of Dlx3 may interfere with GR, resulting in repression in placental cells versus induction in gonadotrope cells. These results demonstrate the stimulatory role played by glucocorticoids in GSU gene expression in the pituitary gonadotrope, in contrast to repression in placental cells, and highlight the tissue-specific nature of steroid hormone action.