We have previously shown that hypercapnic chemoreflex in prepro-orexin knockout mice (ORX-KO) is attenuated during wake but not sleep periods. In that study, however, hypercapnic stimulation had been chronically applied for 6 hrs because of technical difficulty to change the composition of the inspired gas mixture without distorting the animal's vigilance states. Here we examine possible involvement of orexin in acute respiratory chemoreflex during wake periods. Ventilation was recorded together with electroencephalography and electromyography before and after intracerebroventricular administration of orexin or an orexin receptor antagonist, SB-334867. A hypercapnic (5 or 10% CO₂) or hypoxic (15 or 10% O₂) gas mixture was introduced into the recording chamber for 5 min. Respiratory parameters were analyzed only for quiet wakefulness. When mice breathed normal room air, orexin-A and orexin-B but not vehicle or SB-334867 increased minute ventilation in both ORX-KO and wild-type (WT) mice. As expected, hypercapnic chemoreflex in vehicle-treated ORX-KO mice (0.22±0.03 ml·min^-1·g^-1·% CO₂^-1) was significantly blunted compared with that in WT mice (0.51±0.05 ml·min^-1·g^-1·% CO₂^-1). Supplementation of orexin-A or -B (3 nmol) partially restored the hypercapnic chemoreflex in ORX-KO (0.28±0.03 ml·min^-1·g^-1·% CO₂^-1 for ORX-A and 0.32±0.04 ml·min^-1·g^-1·% CO₂^-1 for ORX-B) mice. In addition, injection of SB-334867 (30 nmol) in WT mice decreased hypercapnic chemoreflex (0.39±0.04 ml·min^-1·g^-1·% CO₂^-1). On the other hand, hypoxic chemoreflex in vehicle-treated ORX-KO and SB-treated WT mice was not different from that in corresponding controls. Our findings suggest that orexin plays a crucial role in CO₂ sensitivity at least during wake periods in mice.