We used the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pHi) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO2/HCO3(-)-free medium buffered to pH 7.40 with HEPES at 37 degrees C, the cells had a mean pHi of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO3(-) caused the steady-state pHi to increase by an average of 0.35, suggesting the presence of a HCO3(-) -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3(-)-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pHi to fall to a value substantially below the initial one. pHi spontaneously recovered from this acid load, stabilizing at a value approximately 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cell bathed in HEPES-buffered solutions, removing extracellular Na+ caused pHi to decrease rapidly by 0.5. Returning the Na+ caused pHi to increase rapidly, indicating the presence of an Na(+)-dependent/HCO3(-)-dependent acid-extrusion mechanism; the final pHi after returning Na+ was approximately 0.08 higher than the initial value. This pHi recovery elicited by returning Na+ was not substantially affected by 50 microM ethylisopropylamiloride (EIPA), but was speeded up by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).(ABSTRACT TRUNCATED AT 250 WORDS)