We used the pH-sensitive dye BCECF to evaluate the effect of acute (5-10 min) hypoxia (approximately 3% O(2)) on the regulation of intracellular pH (pH(i)) in astrocyte populations cultured from rat hippocampus. For cells in the nominal absence of CO(2)/HCO(3)(-) at an extracellular pH of 7.40 (37 degrees C), acute hypoxia caused a small (0.05) decrease in steady-state pH(i), but increased the pH(i) recovery rate from an acid load during all but the late phase of the pH(i) recovery. During such pH(i) recoveries, the total acid extrusion rate (phi(E), the product of dpH(i)/dt and proton buffering power) decreased with increasing pH(i). Hypoxia alkali shifted the plot of phi(E) vs. pH(i); over the upper approximately 85% of the phi(E) range, this shift was 0.15-0.30. Hypoxia also stimulated the pH(i) recovery rate from an alkali load. Under normoxic conditions, switching the extracellular buffer to 5% CO(2)/22 mM HCO(-)(3) also alkali shifted the phi(E)-pH(i) plot (upper approximately 85%) by 0.4-0.5. Superimposing hypoxia on CO(2)/HCO(-)(3) further alkali shifted the phi(E)-pH(i) plot (upper approximately 85% of the phi(E) range) by 0.05-0.15. The SITS-insensitive component of phi(E) was alkali shifted by 0.20-0.30, whereas the SITS-sensitive component of phi(E) was depressed in the low pH(i) range. Thus, in the nominal absence of CO(2)/HCO(3)(-), acute hypoxia has little effect on steady-state pH(i) but stimulates acid extrusion and acid loading, whereas in the presence of CO(2)/HCO(-)(3), hypoxia stimulates the SITS-insensitive but inhibits the SITS-sensitive acid extrusion.