The objective of this study was to assess the influence of Ca 2+ influx on intracellular pH (pH i ) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH i . Since this increase was similar in the presence and nominal absence of HCO 3 − , it probably did not reflect influx of HCO 3 − via a Na + -HCO 3 − symporter. Furthermore, since it occurred in the absence of external Ca 2+ (or a measurable rise in Ca i 2+ ) it seemed independent of Ca 2+ influx. It is tentatively concluded that the rise in pH i was due to reduced passive influx of H + along the electrochemical gradient, which is reduced by depolarization. In Ca 2+ -containing solutions, depolarization led to a rebound increase in pH i above control. This, and the rebound found after glutamate transients, may reflect Ca 2+ -triggered phosphorylation and upregulation of the Na + /H + antiporter which extrudes H + from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca i 2+ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na + from the external solution did not alter the rise in pH i , it was probably not due to accelerated H + extrusion. However, removal of Ca 2+ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca 2+ /2H + exchange across plasma membranes.