The generation of activity-evoked extracellular alkaline shifts has been linked to the presence of external Ca2+ or Ba2+. We further investigated this dependence using pH- and Ca2+-selective microelectrodes in the CA1 area of juvenile, rat hippocampal slices. In HEPES-buffered media, alkaline transients evoked by pressure ejection of RS-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) averaged ∼0.07 unit pH and were calculated to arise from the equivalent net addition of ∼1 mM strong base to the interstitial space. These alkaline responses were correlated with a mean decrease in [Ca2+]o of ∼300 μM. The alkalinizations were abolished reversibly in zero-Ca2+ media, becoming indiscernible at a [Ca2+]o of 117 ± 29 μM. Addition of as little as 30–50 μM Ba2+ caused the reappearance of an alkaline response. In approximately one-fourth of slices, a persistent alkaline shift of ∼0.03 unit pH was observed in zero-Ca2+ saline containing EGTA. In HEPES media, addition of 300 μM Cd2+, 100 μM Ni2+, or 100 μM nimodipine inhibited the alkaline shifts by roughly one-half, one-third, and one-third, respectively, whereas Cd+ and Ni2+ in combination fully blocked the response. In bicarbonate media, by contrast, Cd+ and Ni2+blocked only two-thirds of the response. In the presence of bicarbonate, Ni2+ caused an unexpected enhancement of the alkalinization by ∼150%. However, when the extracellular carbonic anhydrase was blocked by benzolamide, addition of Ni2+reduced the alkaline shift. These results suggested that Ni2+ partially inhibited the interstitial carbonic anhydrase and thereby increased the alkaline responses. These data indicate that an activity-dependent alkaline shift is largely dependent on the entry of Ca2+ or Ba2+ via voltage-gated calcium channels. However, sizable alkaline transients still can be generated with little or no external presence of these ions. Implications for the mechanism of the activity-dependent alkaline shift are discussed.