Corticotropin releasing factor (CRF) plays a central role in neuroendocrine, autonomic, immune, and behavioral responses to stressors. We analyzed the proximal promoters of two Xenopus laevis CRF genes and found them to be remarkably conserved with mammalian CRF genes. We found several conserved cis elements in the frog CRF genes including a cyclic AMP response element (CRE), activator protein 1 binding sites (AP1s), and glucocorticoid response elements (GREs). Exposure to a physical stressor caused a rapid elevation in phosphorylated CRE binding protein (pCREB; 20 min) and CRF (1 h) in the anterior preoptic area of juvenile frogs. CREB bound to the putative frog CREs in vitro, which was disrupted by point mutations introduced into the CRE. The frog proximal CRF promoters supported basal transcription in transfection assays, and forskolin caused robust transcriptional activation. Mutagenesis of the CRE or overexpression of a dominant-negative CREB reduced forskolin-induced promoter activation. Using electroporation-mediated gene transfer in tadpole brain, we show that the proximal CRF promoters support cyclic AMP or stressor-dependent transcription in vivo, which was abolished by mutation of the CRE. Using chromatin immunoprecipitation we found that CREB associated with the proximal frog CRF promoter in vivo in a stressor-dependent manner. These data provide strong support for the hypothesis that stressor-induced CRF gene activation in vivo depends on CREB binding to the CRE in the promoter. Our findings show that the basic regulatory elements of the CRF gene responsible for stressor-induced activation arose early in vertebrate evolution and have been maintained by strong positive selection.