The ventilatory response to acute hypoxia in mammalian species is biphasic, an initial hyperventilatory response is followed by a reduction in ventilation within 2-3 min below the peak level (roll-off). Brain amino acid neurotransmitters also change during hypoxia. This study explores the role of neurotransmitters in anesthetized adult Sprague Dawley rats mechanically ventilated during 20 min of 10% O2 breathing. Phrenic nerve activity was recorded, and microdialysate concentrations of selected amino acids were determined at 3- to 5-min intervals in respiratory chemosensitive areas of the ventrolateral medulla (VMS) 1.25-2.00 mm below the surface. Phrenic nerve output was biphasic during hypoxia, concurrent with a rapid glutamate and gradual GABA increase. Taurine first decreased, then increased. In both intact and chemodenervated animals, time-dependent change in phrenic nerve activity during hypoxia was associated with corresponding changes in glutamate, GABA, and taurine concentrations, suggesting that cumulative effects of changes in the concentration of these three amino acids could account for response of the phrenic nerve to hypoxia.