Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue pH- CO2/H+ sensors located in the brainstem respond to changes in pH to regulate depth and frequency of breathing. A region of the caudal brainstem called the retrotrapezoid nucleus (RTN) is thought to be an important site of chemoreception; it contains a population of neurons that are highly sensitive to pH changes in vivo and in vitro, are glutamatergic and project directly respiratory centers to influence breathing(Mulkey et al., 2004). The mechanism by which chemoreceptors sense pH involves inhibition of an unidentified background K+ conductance(Mulkey et al., 2004; Mulkey et al., 2007). There is also evidence that ATP is an important paracrine modulator of chemoreception(Gourine et al., 2005). For example, previous in vivo studies showed that hypercapnia caused discrete release of ATP within the RTN(Gourine et al., 2005). In addition, application of ATP into the RTN stimulated respiratory output, whereas application of an ATP receptor antagonist (pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate, PPADS) to the same region decreased CO2 respiratory sensitivity(Gourine et al., 2005). Further, we showed in vitro that ATP can activate pH-sensitive neurons by a P2Y receptor-dependent mechanism, but that pH-sensitivity of RTN neurons was retained in the presence of PPADS(Mulkey et al., 2006). Together, these results suggest that RTN neurons are intrinsically pH-sensitive but that CO2-evoked release of ATP can modulate the activity of pH-sensitive neurons and contribute to respiratory drive. The source of ATP released in the RTN during hypercapnia is poorly understood. In other brain regions, ATP is a common glialtransmitter and observations first made three decades ago show that a subset of non-spiking RTN cells are CO2/H+-sensitive (Fukuda et al., 1978), suggesting that these pH-sensitive glia could be the source of ATP during hypercapnia. However, the identity of these pH-sensitive glial cells, the mechanism by which they sense changes in pH and their potential contribution to respiratory drive remain unknown. To address these questions, we use the brain slice preparation and a combination of patch-clamp electrophysiology and immunohistochemistry to identity pH-sensitive glia and determine the mechanism by which they sense pH. We found that ~20% of RTN glia are pH-sensitive and of these 86% were immunoreactive for aldehyde dehydrogenase1L1, a selective marker of protoplasmic astrocytes. In current clamp, pH-sensitive RTN astrocytes have a resting membrane potential of -80 ± 1 mV and respond to acidification from pH 7.5 to 6.9 with a depolarization of 8.8 ± 1.4 mV. This H+ response was mimicked by 100 μM Ba2+ (7.9 ± 3.0 mV), suggesting that an inwardly rectifying K+ channel contributes to pH-sensitivity of these cells. In voltage clamp (-80 mV holding potential, 0.5 μM TTX), pH-sensitive astrocytes respond to bath alkalization from 7.3 to 7.5 with increased outward current of 48.6 ± 14.1 pA, whereas acidification to 6.9 decreased outward current by 29.1 ± 5.9 pA. The pH-sensitive current was inhibited 96.5 ± 3.6% by 100 μM Ba2+ and 72.7 ± 21.3% by 100 μM desipramine (a specific Kir4.1 antagonist; Su et al., 2007), suggesting that heteromeric Kir4.1-Kir5.1 channels confers pH-sensitivity to these astrocytes. To test the hypothesis that pH-sensitive RTN astrocytes stimulate activity of pH-sensitive neurons by a purinergic mechanism, we determine the effect of fluorocitrate-mediated astrocyte activation on pH-sensitive neurons under control conditions and in the presence of PPADS. Exposure to fluorocitrate (100 µM) increased neuronal activity by 1.7 ± 0.2 Hz and increased pH-sensitivity ~40%. The effects of fluorocitrate on pH-sensitive neurons were blocked with PPADS (100 µM). However, in the absence of fluorocitrate PPADS had no affect on neuronal pH-sensitivity, suggesting that in the slice preparation H+ alone is unable to elicit ATP release from astrocytes. To the extent that fluorocitrate selectively affects astrocytes (Willoughby et al., 2003), these results suggest that astrocytes can release ATP and activate pH-sensitive neurons, presumably by a P2Y-dependent mechanism(Mulkey et al., 2006), to increase chemoreceptor output. Together, these results strongly suggest that pH-sensitive RTN glia are protoplasmic astrocytes that sense H+ by inhibition of heteromeric Kir4.1-Kir5.1 channels and contribute to chemoreception by a purinergic-dependent mechanism.