The effects of extracellular pH (pHo) on calcium-sensing non-selective cation (csNSC) channels in cultured mouse hippocampal neurons were investigated using whole-cell voltage-clamp and current-clamp recordings. Decreasing extracellular Ca2+ concentrations ([Ca2+]o) activated slow and sustained inward currents through the csNSC channels. Decreasing pHo activated amiloride-sensitive transient proton-gated currents which decayed to baseline in several seconds. With proton-gated channels inactivated by pre-perfusion with low pH solution or blocked by amiloride, decreasing pHo to 6.5 inhibited the csNSC currents with a leftward shift of the Ca2+ dose-inhibition curve. Increasing pH to 8.5, on the other hand, caused a rightward shift of the Ca2+ dose-inhibition curve and potentiated the csNSC currents. Intracellular alkalinization following bath perfusion of quinine mimicked the potentiation of the csNSC currents by increasing pHo, while intracellular acidification by addition and subsequent withdrawal of NH4Cl mimicked the inhibition of the csNSC currents by decreasing pHo. Intracellular pH (pHi) imaging demonstrated that decreasing pHo induced a corresponding decrease in pHi. Including 30 mM Hepes in the pipette solution eliminated the effects of quinine and NH4Cl on the csNSC currents, but only partially reduced the effect of lowering pHo. In current-clamp recordings, decreasing [Ca2+]o induced sustained membrane depolarization and excitation of hippocampal neurons. Decreasing pHo to 6.5 inhibited the low [Ca2+]o-induced csNSC channel-mediated membrane depolarization and the excitation of neurons. Our results indicate that acidosis may inhibit low [Ca2+]o-induced neuronal excitation by inhibiting the activity of the csNSC channels. Both the extracellular and the intracellular sites are involved in the proton modulation of the csNSC channels.