Two newly identified, overlapping (1 bp) glucocorticoid response elements (GREs) at −759 and −773 bp in the promoter of the rat phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) gene are primarily responsible for its glucocorticoid sensitivity, rather than the originally identified −533-bp GRE. A dose-dependent increase in PNMT promoter activity was observed in RS1 cells transfected with a wild-type PNMTpromoter-luciferase reporter gene construct and treated with dexamethasone (maximum activation at 0.1 μM). The type II glucocorticoid receptor antagonist RU38486 (10 μM) fully inhibited dexamethasone (1 μM) activation of the PNMT promoter, consistent with classical glucocorticoid receptors mediating corticosteroid-stimulated transcriptional activity. Relative IC₅₀ values from gel mobility shift competition assays showed that the −759-bp GRE has a 2-fold greater affinity for the glucocorticoid receptor than the −773-bp GRE. Site-directed mutation of the −533-, −759-, and −773-bp GREs alone or in tandem demonstrated that the −759-bp GRE was also functionally more important, but both the −759- and −773-bp GREs are required for maximum glucocorticoid responses. Moreover, the −533-bp GRE, rather than increasing glucocorticoid sensitivity of the promoter, may limit corticosteroid responsiveness mediated via the −759- and −773-bp GREs. Finally, the glucocorticoid receptor bound to the −759- and −773-bp GREs interacts cooperatively with Egr-1 and/or AP-2 to stimulate PNMT promoter activity in RS1 cells treated with dexamethasone. In contrast, glucocorticoid receptors bound to the −533-bp GRE only seem to participate in synergistic activation of thePNMT promoter through interaction with activator protein 2.