The hypothalamic-pituitary-adrenal axis regulates mammalian stress responses by secreting glucocorticoids. The magnitude of the response is in part determined by gender, for in response to a given stressor, circulating glucocorticoids reach higher levels in female rats than in males. This gender difference could result from estrogen regulation of the corticotropin-releasing hormone (CRH) promoter via either of its receptors: estrogen receptor (ER)alpha or ERbeta. Immunocytochemistry revealed that a subset (12%) of medial parvocellular CRH neurons in the rat hypothalamus contain ERbeta but not ERalpha. To determine whether ERs could regulate CRH promoter activity, we cotransfected cells with a CRH promoter construct and either ERalpha or individual ERbeta isoforms. ERalpha weakly stimulated CRH promoter transcriptional activity in a ligand-independent manner. Conversely, all ERbeta isoforms tested stimulated CRH promoter activity with different ligand profiles. ERbeta1 and ERbeta2delta3 displayed constitutive activity (ERbeta1 more than ERbeta2delta3). Ligand-dependent activity of beta isoforms 1 and 2 was altered by an Exon3 splice variant (delta3) or by the additional 18 amino acids in the ligand-binding domain of ERbeta2 isoforms. Lastly, we suggest that ER regulation of CRH takes place through an alternate pathway, one that requires protein-protein interactions with other transcription factors or their associated complexes. However, a pure ER-activator protein-1 alternate pathway does not appear to be involved.