Glucocorticoids (GCs) exert feedback regulation on corticotropin-releasing factor (CRF) neurons in mammals. The nature of GC actions is cell-type specific, being either inhibitory (e.g. paraventricular nucleus) or stimulatory (e.g. amygdala and bed nucleus of the stria terminalis). Nothing is known about differential regulation of CRF gene expression by GCs in nonmammalian vertebrates. We studied the actions of GCs on CRF expression in discrete brain regions of the frog Xenopus laevis. Treatment with corticosterone (CORT) decreased, whereas the corticosteroid synthesis inhibitor metyrapone increased CRF expression in the anterior preoptic area (homolog of the mammalian paraventricular nucleus), as measured by CRF primary transcript, mRNA, and CRF immunoreactivity (ir) (by immunocytochemistry). By contrast to the preoptic area, CORT increased CRF-ir in the medial amygdala and bed nucleus of the stria terminalis, whereas metyrapone decreased CRF-ir in the medial amygdala. CRF-ir and glucocorticoid receptor-ir were colocalized in cells in the frog brain. In transient transfection assays in PC-12 cells, GCs decreased forskolin-induced activation of the frog CRF promoters. Treatment with CORT also reduced CRF promoter activity in transfected tadpole brain in vivo. Frog glucocorticoid receptor bound with high-affinity in vitro to regions in the proximal promoters of frog CRF genes that are homologous with the human CRF gene. Our findings suggest that the neural cell-type specificity and molecular mechanisms of GC-dependent regulation of CRF are phylogenetically ancient, and that the limbic pathways mediating behavioral and physiological responses to stressors were likely present in the earliest land-dwelling vertebrates.