The mechanism involved in N-methyl-D-glucamine (NMDA)-induced Ca²⁺-dependent intracellular acidosis is not clear. In this study, we investigated in detail several possible mechanisms using cultured rat cerebellar granule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM]. When 100 µM NMDA or 40 mM KCl was added, a marked increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and a decrease in the intracellular pH were seen. Acidosis was completely prevented by the use of Ca²⁺-free medium or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following four mechanisms that could conceivably have been involved were excluded: 1) Ca²⁺ displacement of intracellular H⁺ from common binding sites; 2) activation of an acid loader or inhibition of acid extruders; 3) overproduction of CO₂ or lactate; and 4) collapse of the mitochondrial membrane potential due to Ca²⁺ uptake, resulting in inhibition of cytosolic H⁺ uptake. However, NMDA/KCl-induced acidosis was largely prevented by glycolytic inhibitors (iodoacetate or deoxyglucose in glucose-free medium) or by inhibitors of the Ca²⁺-ATPase (i.e., Ca²⁺/H⁺ exchanger), including La³⁺, orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPase is involved in NMDA-induced intracellular acidosis in granule cells. We also provide new evidence that NMDA-evoked intracellular acidosis probably serves as a negative feedback signal, probably with the acidification itself inhibiting the NMDA-induced [Ca²⁺]_i increase.