Accumulating evidence shows possible participation of orexin, a hypothalamic neuropeptide also known as hypocretin, in regulation of breathing. In this study we examined whether hypercapnic stimulation in vivo activates orexin-containing neurons in the hypothalamus. A hypercapnic gas mixture of 10% CO(2), 21% O(2), and residual N(2) or room air was inhaled to awake and freely moving mice for 3h. Their brains were examined by double immunohistochemical staining for orexin and c-Fos. We found that inhalation of CO(2) significantly increased c-Fos expression in the orexin-containing cells that were located in the perifornical region and the dorsomedial hypothalamus (15.8+/-1.6% for room air vs. 28.4+/-2.7% for CO(2), p=0.011) but not in the lateral hypothalamic area (12.3+/-1.2% for room air vs. 12.4+/-3.0% for CO(2)). Although current methodology cannot differentiate between direct effect of CO(2) on the orexin-containing neurons and indirect one through other neurons, this is the first report showing that inhalation of CO(2) did activate the orexin-containing neurons in vivo.