Cerebral blood flow is highly sensitive to changes in CO2/H+, where an increase in CO2/H+ causes vasodilation and increased blood flow. Tissue CO2/H+ also functions as the main stimulus for breathing by activating chemosensitive neurons in brainstem respiratory centers such as the retrotrapezoid nucleus (RTN). Interestingly, RTN astrocytes also support chemoreception by providing a CO2/H+-dependent purinergic drive that enhances activity of chemosensitive neurons. Considering that CO2/H+-induced vasodilation would accelerate removal of CO2/H+ and potentially counteract the drive to breathe, we hypothesize that chemosensitive brain regions have adapted a means of preventing vascular CO2/H+ reactivity, that may also be mediated via astrocyte purinergic signalling. We measured changes in arteriole diameter in response to CO2/H+ in control conditions and in the presence of the ATP receptor (P2R) antagonist suramin (100µM), in adult rat brainstem and cortical slice preparations. All procedures were performed in accordance with the National Institute of Health, University of Connecticut and University São Paulo Animal Care and Use Guidelines. We also recorded diameter changes during bath application of t-ACPD (to elicit Ca2+ elevations in astrocytes; 50µM). Consistent with our hypothesis, we found that exposure to CO2/H+ or t-ACPD under control conditions caused a small yet significant decrease in arteriole diameter, but when purinergic receptors were inhibited, exposure to CO2/H+ had no effect on arteriole diameter. As expected, cortical arterioles responded to CO2/H+ by vasodilation under control conditions. We also recorded changes in pial vessel diameter in vivo in response to increased CO2/H+ alone or in the presence of the P2R blocker PPADS. Consistent with our in vitro data, we found that breathing high CO2 constricted ventral medullary surface (VMS) vessels by 13 ± 2%. However, when P2R’s are blocked, increasing inspired CO2 caused a 10 ± 1% vasodilatation. Furthermore, application of PPADS to the RTN decreased the ventilatory response to CO2 and we found that localized application of a vasoconstrictor (phenylephrine; 1µM) and vasodilator (nitroprusside; 1 µM) to the VMS increased and decreased ventilatory responses to CO2, respectively. Our data shows that purinergic signaling in the retrotrapezoid nucleus (RTN) maintains vascular tone during high CO2/H+ and disruption of this mechanism decreases the ventilatory response to CO2. This discovery that CO2/H+ -dependent regulation of vascular tone in the RTN is the opposite of that of the rest of the cerebral vascular tree is novel and fundamentally important for understanding how regulation of vascular tone impacts localized neural function and the drive to breathe.