Purinergic signalling mediated by ATP has been associated with central chemosensory function. A decrease in pH from 7.4 to 7.2 induces ATP release (peak 1.0±0.3 μM, n=8) from the VMS as detected using ATP biosensors placed on the brainstem surface in horizontal slices prepared from young adult rats. Using in vitro preparations and novel genetically encoded Ca2+ indicator based on cyclically permutated GFP – Case 12 (Souslova et al.2007) targeted to astrocytes with a cell-specific adenoviral vector, we show that astrocytes in the ventral regions of the medulla oblongata are highly sensitive to changes in pH. All experiments were performed on Sprague-Dawley rats (University College London breeding colony, London, UK) in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Decrease in extracellular pH from 7.4 to 7.2 induces transient increases in [Ca2+]i in astrocytes from dissociated neuro-glial cultures prepared from the VMS, in ventral astrocytes of organotypic brainstem slice cultures and in acute horizontal brainstem slices of adult rats. Increases in [Ca2+]i in ventral astrocytes evoked by lowering bath pH is blocked in the presence of the ATP hydrolyzing enzyme apyrase (25 U ml-1). ATP receptor antagonists such as MRS2179 (3 μM), PPADS (5 μM) and TNP-ATP (20 nM) effectively reduce [Ca2+]i responses evoked by lowering pH in VMS astrocytes by 82%, 80% , and 83%, respectively. This pharmacological profile suggests the involvement of ionotropic P2X receptors. Indeed, acidification-induced [Ca2+]i responses in astrocytes were absent in Ca2+-free medium (with an addition of 2 mM Mg2+ and 1 mM EGTA to chelate any residual Ca2+), indicating that a decrease in pH triggers Ca2+ entry from the extracellular space. Furthermore, VMS astrocytes loaded with Fura-2 AM and incubated in the medium containing 50 μM Mn2+ showed an immediate decrease in fluorescence in response to ATP. These data directly demonstrate opening of Ca2+-permeable channels. Acidification-induced [Ca2+]i responses in VMS astrocytes were not affected by either carbenoxolone (10 μM, that blocks pannexin hemichannels in this concentration) or lanthanum (La3+; 100 μM – a connexin hemichannel blocker that does not affect gap junctions). Inhibitors of vesicular transport brefeldine A and bafilomycin A both effectively abolished [Ca2+]i excitation of VMS astrocytes evoked by lowering external pH. Incubation of astrocytes with Mant-ATP, a fluorescent nucleotide analogue used for studying ATP stores and vesicular transport, labelled populations of vesicles. Acidification of external medium induced rapid destaining of the Mant-ATP loaded vesicles. These data demonstrate that in VMS astrocytes acidification of external medium leads to Ca2+ entry and vesicular release of ATP to propagate Ca2+ excitation across the astrocytic network.