We used the pH-sensitive fluorescent dye BCECF to study intracellular pH (pHi) regulation in primary cultures of rat astrocytes and C6 glioma cells. Both cell types contain three pH-regulating transporters: (1) alkalinizing Na+/H+ exchange; (2) alkalinizing Na+ + HCO3 −/Cl−exchange; and (3) acidifying Cl−/HCO3− exchange. Na+/H+ exchange was most evident in the absence of CO2; recovery from acidification was Na+ dependent and amiloride sensitive. Exposure to CO2 caused a cell alkalinization that was inhibited by DIDS, dependent on external Na+, and inhibited 75% in the absence of Cl− (thus mediated by Na+ + HCO3−/Cl− exchange). When pHi was increased above the normal steady-state pHi, a DIDS-inhibitable and Na+ -independent acidifying recovery was evident, indicating the presence of Cl− /HCO3−exchange. Astrocytes, but not C6 cells, contain a fourth pH-regulating transporter, Na+ −HCO3− cotransport; in the presence of CO2, depolarization caused an alkalinization of 0.12 +− 0.01 (n = 8) and increased the rate of CO2-induced alkalinization from 0.23 ± 0.02 to 0.42 ± 0.03 pH unit/min. Since C6 cells lack the Na+ -HCO3+ cotransporter, they are an inferior model of pHi regulation in glia. Our results differ from previous observations in glia in that: (1) Na+ /H+ exchange was entirely inhibited by amiloride; (2) Na+ + HCO3−/Cl− exchange was present and largely responsible for CO2−induced alkalinization; (3) Cl− /HCO3− exchange was only active at pHi values above steady state; and (4) depolarization-induced alkalinization of astrocytes was seen only in the presence of CO2.