Ventral regions of the medulla oblongata of the brainstem are populated by astrocytes sensitive to physiological changes in P_CO2/[H⁺]. These astrocytes respond to decreases in pH with elevations in intracellular Ca²⁺ and facilitated exocytosis of ATP-containing vesicles. Released ATP propagates Ca²⁺ excitation among neighboring astrocytes and activates neurons of the brainstem respiratory network triggering adaptive increases in breathing. The mechanisms linking increases in extracellular and/or intracellular P_CO2/[H⁺] with Ca²⁺ responses in chemosensitive astrocytes remain unknown. Fluorescent imaging of changes in [Na⁺]_i and/or [Ca²⁺]_i in individual astrocytes was performed in organotypic brainstem slice cultures and acute brainstem slices of adult rats. It was found that astroglial [Ca²⁺]_i responses triggered by decreases in pH are preceded by Na⁺ entry, markedly reduced by inhibition of Na⁺/HCO₃^- cotransport (NBC) or Na⁺/Ca²⁺ exchange (NCX), and abolished in Na⁺-free medium or by combined NBC/NCX blockade. Acidification-induced [Ca²⁺]_i responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na⁺/HCO₃^- cotransporter NBCe1. Sensitivity of astrocytes to changes in pH was not affected by inhibition of Na⁺/H⁺ exchange or blockade of phospholipase C. These results suggest that in pH-sensitive astrocytes, acidification activates NBCe1, which brings Na⁺ inside the cell. Raising [Na⁺]_i activates NCX to operate in a reverse mode, leading to Ca²⁺ entry followed by activation of downstream signaling pathways. Coupled NBC and NCX activities are, therefore, suggested to be responsible for functional CO₂/H⁺ sensitivity of astrocytes that contribute to homeostatic regulation of brain parenchymal pH and control of breathing.Significance statementBrainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain P_CO2/[H⁺]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na⁺/HCO₃^- cotransport, which brings Na⁺ inside the cell. Raising [Na⁺]_i activates the Na⁺/Ca²⁺ exchanger to operate in a reverse mode leading to Ca²⁺ entry. This identifies a plausible mechanism of functional CO₂/H⁺ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.