Intracellular pH (pH,) and the mechanisms of pH, regulation have been investigated in cultured microglial cells from mouse brain using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). Cells were acidified by a pulse of NH,+ (4-5 min; 20 mM) and the subsequent pH, recovery from an acidification was studied. In HC0,--free saline, pH regulation was dependent on extracellular "a+] and sensitive to amiloride, indicating the involvement of the Na+/H+ exchanger. In HC0,--containing solution 2 mM amiloride slowed but did not block pH, recovery; the recovery however was dependent on extracellular "a+] and sensitive to 0.3 mM DIDS, suggesting the presence of Na+/HCO, cotransporter and/or Na + -dependent Cl-/HCO,-exchanger. The involvement of a Na-dependent C1-/ HCO,-exchanger was inferred from the observation that removal of C1-or application of 1 mM furosemide decreased but did not block the recovery rate. Increasing [K+], resulted in an alkalinization by a process that was neither HC0,-nor Na+-dependent, nor DIDS-and amiloride-inhibitable. In conclusion, microglial cells express a distinct set of pH regulatory carriers which control for a defined level of pHi. An increase in [K+I0 can offset this level.