Increasing lines of evidence indicate that estrogen acts as a neuroprotective agent through a nongenomic mechanism. We tested the hypothesis that 17h-estradiol could rapidly attenuate glutamate-induced calcium (Ca2+) overload in rat primary hippocampal neurons via a membrane receptor-dependent mechanism. The bulk cytosolic intracellular Ca2+ level was measured in neurons with fluorescent Ca2+ probe fluo3. Preexposure of primary cultured hippocampal neurons to 17h-estradiol for 3 min attenuated intracellular Ca2+ increase induced by glutamate in a concentration-dependent manner. The action of 17h-estradiol was reversible after washout. Administration of membraneimpermeable 17h-estradiol conjugated to bovine serum albumin (E2–BSA) produced the same effect, suggesting possible involvement of cell membrane receptors. ICI 182,780, a specific estrogen receptor (ER) antagonist, blocked the neuronal response to 17h-estradiol and estradiol BSA, indicating a role of specific ERs. The present study demonstrates that 17h-estradiol acutely reduces glutamate-stimulated intracellular Ca2+ increase via ERs probably on the cell surface of the hippocampal neurons.