The expression of the gene encoding the rat beta 1-adrenergic receptor is suppressed by glucocorticoids (Kiely et al., 1994). Within the 3.2-kb 5'-flanking region of the promoter, two potential glucocorticoid response elements (GREs) at -950 and -2791 relative to the translational ATG were identified. Characterization of the glucocorticoid-responsive sequences in the 5'-flanking region of the beta 1-adrenergic receptor gene was explored in rat C6 glioma cells and human HepG2 hepatoma cells using transient expression of beta 1-adrenergic receptor-luciferase fusion genes. The ability of glucocorticoids to suppress luciferase expression was not altered when the most 5'-localized GRE was deleted. Deleting the potential GRE at -950, in contrast, abolished glucocorticoid-induced suppression of the beta 1-adrenergic receptor-luciferase gene transcription. A 25-bp element containing the GRE sequence between nucleotides -950 and -926 confers glucocorticoid-dependent inhibition of transcription to a neutral promoter. Gel mobility shift assays with the alpha-subunit of the human glucocorticoid receptor (hGR alpha) expressed in reticulocyte lysates demonstrated specific binding to the 25-bp sequence harboring the putative GRE. We report an inhibitory GRE in the promoter of the rat beta 1-adrenergic receptor gene that is conserved among the rat, human, and mouse genes.