We compared the hypoxic ventilatory response (HVR) of two groups of unrestrained awake male rats (300-550 g): those acclimatized to hypoxia > 7 wk at simulated altitude (380 Torr, n = 12) and sea level controls (n = 8). Chronic catheters were placed in the iliac artery and vein 3-7 days before study. An "on-line" system was used to measure arterial PO2 and PCO2. Arterial blood was drawn via a roller pump past O2 and CO2 electrodes and returned to the vein. Batch samples were taken before and after HVR measurements for calibrating and determining arterial pH and hematocrit. Inspired ventilation, tidal volume, and respiratory frequency were measured with barometric pressure plethysmography at several levels of inspired O2 fraction (0.08-0.30) maintained for 15 min. For isocapnic HVR, inspired CO2 fraction was increased as necessary to maintain arterial PCO2 at the hyperoxic level. In both groups, poikilocapnic HVRs (inspired CO2 fraction = 0) were significantly less than isocapnic HVRs. Isocapnic HVRs were significantly greater in hypoxia-acclimatized (2,783 +/- 233 ml.min-1.kg-1) than in sea level control rats (1,826 +/- 106 ml.min-1.kg-1), mainly due to a significant increase in tidal volume (P < 0.05). In conclusion, relieving hypocapnia in hypoxia, by maintaining isocapnia, reveals a significant increase in the ventilatory response to arterial PO2 in awake rats with chronic hypoxia.