We investigated whether resting ventilation and the hypercapnic ventilatory response vary with time of day in awake adult rats. Respiratory frequency (fR), tidal volume (VT), inspired ventilation (VI), inspiratory interval (tI), carbon dioxide production (VCO2) and abdominal temperature (Tb) were measured before and during a hypercapnic stimulus (3.5% CO2 in air) at 10:00 and 22:00 h. VCO2, Tb and mean inspiratory air flow (VT/tI) were significantly higher at 22:00 h in air. VI/VCO2 was similar at 10:00 and 22:00 h. VI was significantly elevated by hypercapnia and the response at 22:00 h was 2.3 times greater than that at 10:00 h. VT/tI was unchanged at 10:00 h but significantly increased by hypercapnia at 22:00 h. VCO2 was significantly depressed at 10:00 h but not at 22:00 h. Tb was unaffected by hypercapnia. We conclude that the metabolic and ventilatory responses to hypercapnia are dependent on the time of day.