We examined pH regulation in two chemosensitive areas of the brain, the retrotrapezoid nucleus (RTN) and the nucleus tractus solitarius (NTS) to identify the proton transporters involved in regulation of pHi in medullary glia. Transverse brain slices from young rats (P8-P20) were loaded with the pH sensitive probe, BCECF after kainic acid treatment removed neurons. Cells were alkalinized when they were depolarized (extracellular K⁺ increased from 6.24 to 21.24 mM) in the RTN, but not in the NTS. This alkaline shift was inhibited by 0.5 mM DIDS. Removal of CO₂/HCO₃^- or Na⁺ from the perfusate acidified the glial cells, but the acidification after Na⁺ removal was greater in the RTN than in the NTS. Treatment of the slice with EIPA (100 µM) in saline containing CO₂/HCO₃^- acidified the cells in both nuclei, but the acidification was greater in the NTS. Restoration of extracellular Cl^- after Cl^- depletion during the control condition acidified the cells. Immunohistochemical studies of glial fibrillary acid protein demonstrated much denser staining in the RTN compared to the NTS. We conclude that there is evidence of sodium bicarbonate cotransport and sodium hydrogen exchange in glia in the RTN and NTS, but the distribution of glia and the distribution of these pH regulatory functions is not identical in the NTS and RTN. The differential strength of glial pH regulatory function in the RTN and NTS may also alter CO₂ chemosensory neuronal function at these two chemosensitive sites in the brainstem.