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Intracellular pH (pH1) and the mechanisms of pH1 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 NH4+ (4‐5 min; 20 mM) and the subsequent pH1 recovery from an acidification was studied. In HCO3‐‐free saline, pH regulation was dependent on extracellular [Na+] and sensitive to amiloride, indicating the involvement of the Na+/H+ exchanger. In HCO3‐‐containing solution 2 mM amiloride slowed but did not block pH1 recovery; the recovery however was dependent on extracellular [Na+] and sensitive to 0.3 mM DIDS, suggesting the presence of Na+/HCO3 cotransporter and/or Na+‐dependent Cl‐/HCO3‐ exchanger. The involvement of a Na‐dependent Cl‐/HCO3‐ exchanger was inferred from the observation that removal of Cl‐ or application of 1 mM furosemide decreased but did not block the recovery rate. Increasing [K+]0 resulted in an alkalinization by a process that was neither HCO3‐ 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 pH1. An increase in [K+]0 can offset this level. © 1996 Wiley‐Liss, Inc.