The negative glucocorticoid regulation of CRH gene expression is a critical control element in the hypothalamic-pituitary-adrenal axis. In this study, the molecular mechanisms mediating the glucocorticoid repression of cAMP-induced CRH-reporter expression in AtT-20 cells have been examined. In these cells, dexamethasone decreases forskolin-induced expression of CRH-reporter activity in a dose-dependent manner. This repression is mediated by the glucocorticoid receptor (GR) and does not require ongoing protein synthesis. Several binding sites for the GR DNA-binding domain were identified within the CRH 5'-flanking and 5'-untranslated regions utilizing in vitro DNase I protection assays. These sites were independently mutated and/or deleted. Functional studies in transfected cells suggest that none of the protected DNA sequences mediate the glucocorticoid regulation and that the regulatory element(s) mediating negative glucocorticoid regulation is contained within the CRH DNA sequences from -248 to +4 bp relative to the major transcription initiation site. To further localize the DNA sequence(s) responsive to glucocorticoids, DNA fragments containing various amounts of human CRH 5'flanking sequences were inserted 5' to the SV40 promoter. An 18-bp DNA fragment containing the CRH cAMP-responsive element is sufficient to confer both positive cAMP regulation and glucocorticoid repression of cAMP-stimulated expression to the SV40 promoter. These results suggest that glucocorticoid repression of forskolin-activated CRH-reporter expression in AtT-20 cells occurs via interference with the cAMP-mediated activation of gene expression, possibly via direct or indirect interactions between the GR and the cAMP-responsive element-binding proteins.