Relatively little is known about the mechanisms of pHi regulation in mammalian glial cells. We analyzed pHi regulation in rat hippocampal astrocytes in vitro using the pH-sensitive dye BCECF. All experiments were carried out in CO2/HCO3(-)-free solutions. Recovery from NH4(+)-induced acid loads was strongly dependent on the presence of extracellular Na+ and was inhibited by amiloride and its more specific analog EIPA, indicating the presence of Na(+)-H+ exchange in these cells. Removing bath Na+ or adding amiloride caused resting pHi to shift in the acid direction. Even in the absence of bath Na+ or presence of Na+/H+ inhibitors, however, these astrocytes continued to show significant recovery from acid loads. The mechanism of this amiloride-insensitive and Na(+)-independent pHi recovery process was sought and appeared to be a proton pump. In the absence of Na+, recovery from an acid load was completely blocked by the highly specific blocker of vacuolar-type (v-type) H+ ATPase, bafilomycin A1 (BA1). In normal Na+ containing solutions, exposure to BA1 caused a small acid shift in baseline pHi and slowed recovery rate from NH4(+)-induced acid loads by about 32%. The rate of Na(+)-independent pHi recovery was increased by depolarization with 50 mM [K+] solution, and this effect was rapidly reversible and blocked by BA1. These results indicate that, in CO2/HCO3(-)-free solution, pHi regulation in hippocampal astrocytes was mediated by Na(+)-H+ exchange and by a BA1-inhibitable proton pump. Because the proton pump's activity was influenced by membrane potential, this acid exporting mechanism could contribute to the depolarization-induced alkalinization that is seen in astrocytes. Although v-type H(+)-ATPase had been previously isolated from the brain, this is the first report indicating that it has a role in regulating pHi in brain cells.