The internal pH (pHi) regulatory mechanisms of individual rat cerebellar granule cells maintained in tissue culture have been investigated using the fluorescent indicator BCECF (2,7’bis carboxyethyl 5,6 carboxyfluorescein) and quantitative fluorescence microscopy. The steady-state pH, was estimated as 7.27 ± 0.25 in bicarbonate-buffered media and 7.49 ± 0.35 in HEPES-buffered media. Buffering power was estimated at about 8 mM/pH unit from the peak alkalinization and acidification transients seen on addition and removal of NH4CI. Bicarbonate did not appear to contribute to the buffering power estimated in this way. Following an acid load imposed by the ammonium prepulse technique, pHi recovered to steady-state values with first-order kinetics. Recovery was absolutely dependent upon extracellular sodium and, in about half of the cells tested, bicarbonate ions. In cells that did not require bicarbonate for pHi recovery, amiloride (1 mM) inhibited pHi recovery. Removal of extracellular chloride produced a reversible alkalinization of pHi in a third of the cells studied. This alkalinization persisted even when extracellular sodium had been reduced to zero. Removal of extracellular chloride did not inhibit bicarbonate-dependent pHi recovery following an acid load. These results are best explained by the existence of three independent pHi regulatory mechanisms: Na+/H+ exchange, Na+/HCO3− cotransport and Cl−/HCO3− exchange.