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Modulation of cytosolic calcium ([Ca2+]i) plays a key role in intracellular signalling. In neurones, intracellular Ca2+ transients are involved in the regulation of excitability as well as in synaptic transmission and plasticity. Here, we report that depolarization-induced elevation of [Ca2+]i evokes a prominent fall of intracellular pH (pHi) in voltage-clamped hippocampal pyramidal neurones dialysed with fluorescent indicators of H+ and Ca2+. This acidification is caused by exchange of internal calcium for extracellular protons via a vanadate-and eosin-sensitive plasmalemmal Ca2VH+ pump. In view of the potent neuromodulatory actions of H+, these results raise the possibility that changes in excitability and synaptic plasticity, hitherto solely attributed to Ca2+ transients, may include a significant component mediated by pH shifts.