Peripheral chemoreflex activation with potassium cyanide (KCN) in awake rats or in the working heart brainstem preparation (WHBP) produces: a) a sympathoexcitatory/pressor response; b) bradycardia; and c) an increase in the frequency of breathing. Our major aim was to evaluate neurotransmitters involved in mediating the sympathoexcitatory component of the chemoreflex within the NTS. In previous studies in conscious rats, the reflex bradycardia, but not the pressor response, was reduced by antagonism of either NMDA or P2 receptors within the NTS. The present study evaluated a possible dual role of both purinergic P2 and ionotropic glutamate receptors in the NTS for processing the sympathoexcitatory component (pressor response) of the chemoreflex in awake rats as well as in the WHBP. Simultaneous blockade of ionotropic glutamate receptors and P2 receptors by sequential microinjections of kynurenic acid (KYN, 2 nmol/50 nL) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS, 0.25 nmol/50 nL) into the commissural NTS in awake rats produced a significant reduction in both the pressor (+38±3 vs +8±3 mmHg) and bradycardic responses (-172±18 vs -16±13 bpm; n=13), but no significant changes in the tachypnoea measured using plethysmography (270±30 vs 240±21 cpm, n=7) following chemoreflex activation in awake rats. Control microinjections of saline produced no significant changes in these reflex responses. In WHBP, microinjection of KYN (2 nmol/20 nL) and PPADS (1.6 nmol/20 nL) into the commissural NTS attenuated significantly both the increase in thoracic sympathetic activity (+52±2 vs +17±1 %) and the bradycardic response (-151±17 vs -21±3 bpm) but produced no significant changes in the increase of the frequency of phrenic nerve discharge (+0.24±0.02 vs +0.20±0.02 Hz). The data indicate that combined microinjections of PPADS and KYN into the commissural NTS in both awake rats and the WHBP are required to produce a significant reduction in the sympathoexcitatory response (pressor response) to peripheral chemoreflex activation. We conclude that glutamatergic and purinergic mechanisms are part of the complex neurotransmission system of the sympathoexcitatory component of the chemoreflex at the level of the commissural NTS.