Corticotropin-releasing factor (CRF) is a key player in the regulation of the hypothalamic-pituitary-adrenal axis. We already reported a mouse line in which the Venus (enhanced yellow fluorescent protein) gene was inserted to the translation initiation site of the CRF gene by homologous recombination (CRF-Venus). First, the distribution of Venus-expressing neurons was examined in the paraventricular nucleus of the hypothalamus (PVH) in the present study. Second, the effect of glucocorticoid (GC) was examined to see if it affects the Venus expression in the PVH. In a physiological GC state, 60% of Venus neurons expressed CRF, and 30% of CRF neurons expressed Venus. Following stressless removal of GC, Venus expression and CRF expression were enhanced in the PVH, and most Venus neurons became expressing of CRF. Conversely, expression of Venus and CRF was suppressed by GC. In PVH-Venus neurons, expression of copeptin, a peptide coded within the vasopressin gene, was induced by deprivation of GC, which was suppressed by GC administration. Thus, Venus neurons recapitulated partly the GC-dependent responses of PVH-CRF neurons. We generated another line, CRF-VenusΔneo, by deleting the PGK-neo construct from the CRF-Venus. Venus was expressed more constitutively in the PVH of the CRF-VenusΔneo, and almost all Venus neurons expressed CRF in the physiological GC state. CRF-Venus and CRF-VenusΔneo will be of great benefit for unravelling the physiological regulatory mechanisms of CRF neurons.