Previous studies have shown that exposing hippocampal slices to low osmolarity (πo) or to low extracellular NaCl concentration ([NaCl]o) enhances synaptic transmission and also causes interstitial calcium ([Ca2+]o) to decrease. Reduction of [Ca2+]o suggests cellular uptake and could explain the potentiation of synaptic transmission. We measured intracellular calcium activity ([Ca2+]i) using fluorescent indicator dyes. In CA1 hippocampal pyramidal neurons in tissue slices, lowering πo by ∼70 mOsm caused “resting” [Ca2+]i as well as synaptically or directly stimulated transient increases of calcium activity (Δ[Ca2+]i) to transiently decrease and then to increase. In dissociated cells, lowering πo by ∼70 mOsm caused [Ca2+]i to almost double on average from 83 to 155 nM. The increase of [Ca2+]i was not significantly correlated with hypotonic cell swelling. Isoosmotic (mannitol- or sucrose-substituted) lowering of [NaCl]o, which did not cause cell swelling, also raised [Ca2+]i. Substituting NaCl with choline-Cl or Na-methyl-sulfate did not affect [Ca2+]i. In neurons bathed in calcium-free medium, lowering πo caused a milder increase of [Ca2+]i, which was correlated with cell swelling, but in the absence of external Ca2+, isotonic lowering of [NaCl]o triggered only a brief, transient response. We conclude that decrease of extracellular ionic strength (i.e., in both low πo and low [NaCl]o) causes a net influx of Ca2+ from the extracellular medium whereas cell swelling, or the increase in membrane tension, is a signal for the release of Ca2+ from intracellular stores.