To evaluate whether changes in extracellular glutamate (Glu) levels in the central nervous system could explain the depressed hypoxic ventilatory response in hypothermic neonates, 12 anesthetized, paralyzed, and mechanically ventilated piglets <7 days old were studied. The Glu levels in the nucleus tractus solitarius obtained by microdialysis, minute phrenic output (MPO), O₂ consumption, arterial blood pressure, heart rate, and arterial blood gases were measured in room air and during 15 min of isocapnic hypoxia (inspired O₂ fraction = 0.10) at brain temperatures of 39.0 ± 0.5°C [normothermia (NT)] and 35.0 ± 0.5°C [hypothermia (HT)]. During NT, MPO increased significantly during hypoxia and remained above baseline. However, during HT, there was a marked decrease in MPO during hypoxia (NT vs. HT, P < 0.03). Glu levels increased significantly in hypoxia during NT; however, this increase was eliminated during HT (P < 0.02). A significant linear correlation was observed between the changes in MPO and Glu levels during hypoxia (r = 0.61, P < 0.0001). Changes in pH, arterial PO₂, O₂ consumption, arterial blood pressure, and heart rate during hypoxia were not different between the NT and HT groups. These results suggest that the depressed ventilatory response to hypoxia observed during HT is centrally mediated and in part related to a decrease in Glu concentration in the nucleus tractus solitarius.