It has been suggested that orexin is involved in the ventilatory response to CO2. As the Locus coeruleus (LC) is a chemoreceptor site and expresses an extensive population of orexin receptor 1 (OX1R), we tested the hypothesis that OX1R located in the LC is involved in chemoreception. For the surgical procedures, male Wistar rats were subjected to general anesthesia by i.p. ketamine (100 mg/kg) and xylazine (10 mg/kg). One week later, in unanesthetized rats, we injected SB-334867 (OX1R antagonist, 5 mM ) unilaterally into the LC. Pulmonary ventilation (VE) was recorded in a whole body plethysmograph, together with body temperature (Tb, dataloggers), mean arterial pressure (MAP) and heart rate (fH) in air followed by hypercapnia (7% CO2). Hypercapnia caused an increase in VE, which resulted from increases in respiratory frequency (fR) and tidal volume (Vt), but did not affect MAP, fH or Tb in all groups. SB-334867 caused a significant attenuation (70% reduction), of the hyperventilation induced by hypercapnia (n=9) compared with its vehicle (n=6; P< 0.05). This effect was due to both decreased Vt and f. Basal ventilation, MAP, fH or Tb, were not affected by OX1R antagonism. These results suggest that projections of orexin-containing neurons to the LC contribute via orexin R1 to the hypercapnic chemoreflex control in unanesthetized rats.