New Findings •  What is the central question of this study? The hypothesis that nitric oxide and NMDA glutamate receptor activation modulate cardiovascular responses to the microinjection of methyl ATP into the paraventricular nucleus (PVN) was tested in the present study. •  What is the main finding and its importance? The cardiovascular responses that are evoked by the microinjection of methyl ATP into the PVN involve NO production that promotes glutamate release and a subsequent activation of NMDA glutamate receptors in postsynaptic pre‐autonomic neurons, modulating sympathetic nerve activity. Data show new insights into the role of the ATP–NO–glutamate pathway in the PVN in cardiovascular modulation. We hypothesize that a local ATP–NO–NMDA glutamate receptor interaction in the paraventricular nucleus (PVN) modulates the baseline mean arterial pressure and heart rate in unanaesthetized rats. The microinjection of α,β‐methylene ATP methyl ATP; 0.06, 0.12 and 1.2 nmol (100 nl)−1 into the PVN caused pressor and tachycardiac responses. Cardiovascular responses evoked by methyl ATP 0.12 nmol (100 nl)−1 in the PVN were blocked by pretreatment with the ganglion blocker pentolinium (5 mg kg−1 i.v.). Also, responses to the injection of methyl ATP 0.12 nmol (100 nl)−1 into the PVN were reduced by pretreatment with the selective P2 purinergic receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid 0.5 nmol (100 nl)−1, the neuronal NO synthase inhibitor Nω‐propyl‐l‐arginine 0.04 nmol (100 nl)−1 or the selective NMDA glutamate receptor antagonist LY235959 2 nmol (100 nl)−1. In addition, an injection of the NO donor sodium nitroprusside 27 nmol (100 nl)−1 into the PVN caused similar cardiovascular responses to those observed after methyl ATP, which were blocked by local pretreatment with LY235959. Therefore, the present results suggest that cardiovascular responses evoked by methyl ATP in the PVN involve a local production of NO, which promotes local glutamate release and activation of NMDA receptors that are probably located in pre‐autonomic parvocellular neurons, leading to sympathetic nervous system stimulation.