Changes in heart rate (HR), thoracic sympathetic nerve activity (tSNA) and frequency of phrenic nerve discharge (PND) in response to microinjection of L-glutamate before and after local microinjection of ionotropic or metabotropic glutamate receptors antagonists into the commissural subnucleus of the NTS (comNTS) were investigated. The experiments were performed in an in situ unanesthetized decerebrated working heart-brainstem preparation (WHBP), and the main findings were as follows: (a) microinjection of increasing concentrations of L-glutamate (5, 25, 50, 250 and 500 mM) into the comNTS produced bradycardia, increase in tSNA and reduction in the frequency of the PND in a concentration-dependent manner; (b) both bradycardia and increase in tSNA were almost abolished by kynurenic acid (KYN, 250 mM, a nonselective ionotropic glutamate receptor antagonist); (c) the reduction in the frequency of the PND was reversed to an increase in the frequency of the PND after KYN and this increase was blocked by the sequential microinjection of MCPG (100 mM, a nonselective metabotropic glutamate receptor antagonist); and (d) microinjection of increasing concentrations of trans-ACPD (0.5, 1.0, 2.5, 5.0 and 10 mM, a metabotropic glutamate receptor agonist), elicited bradycardia and increase in the frequency of the PND in a concentration-dependent manner, which were blocked by MCPG. Taken together, these data indicate that l-glutamate and its ionotropic receptors are involved in the sympathoexcitatory, bradycardic and reduction in the frequency of the PND responses whereas/although its metabotropic receptors are involved in the bradycardic and mainly in the increase in the frequency of the PND to microinjection of L-glutamate into the comNTS in the WHBP.