We studied the regulation of intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pHi was 7.00 +/- 0.02 in HCO3(-)-containing solutions at an extracellular pH of 7.35. Addition of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or amiloride decreased pHi, as did removal of extracellular Na+, while removal of extracellular Cl- was followed by an increase in pHi. Following exposure to an acid transient induced by increasing the CO2 content from 5 to 15%, pHi rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min-1 (corresponding to a mean acid extrusion rate of 6.3 +/- 0.36 mmolo l-1 min-1). Regulation of pHi was impaired when either amiloride or DIDS was added or Cl- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pHi regulation was completely blocked in the absence of extracellular Na+. The rapid regulation of pHi normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na+, but was partially inhibited by DIDS and by the absence of extracellular Cl-. The results are compatible with the presence of at least three different pHi-regulating mechanisms: a Na+/H+ antiporter, a Na(+)-dependent HCO3-/Cl- exchanger (both regulating pHi during a transient acidification), and a passive Cl-/HCO3- exchanger (regulating pHi during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na+/HCO3- symporter.