Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H+‐mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO2‐evoked ATP release. However, it is not clear whether purinergic signalling also influences CO2/H+‐ responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO2/H+‐sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO2‐responsiveness in vitro and in vivo. In brain slices, cell‐attached recordings of membrane potential show that CO2/H+‐sensitive NTS neurons are activated by focal ATP application; however, P2‐receptor blockade did not affect their CO2/H+‐responsiveness. CO2/H+‐sensitive raphe neurons were unaffected by ATP or P2‐receptor blockade. In vivo, ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2‐receptor into this region no effect on baseline breathing or CO2/H+‐responsiveness. Injections of ATP or a P2‐receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2‐receptor‐dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception. This article is protected by copyright. All rights reserved