Appreciating the physiology of astrocytes and their role in brain functions requires an understanding of molecules that activate these cells. Estradiol may influence astrocyte functions. We now report that estrogen altered intracellular calcium concentration ([Ca(2+)](i)) in neonatal astrocytes that expressed estrogen receptor (ER) mRNA in vitro. Western blotting revealed both ERalpha and ERbeta proteins in both the nuclear fractions and plasma-membrane fractions. Application of 17beta-estradiol (20 nm) to fura 2-loaded astrocytes in vitro stimulated [Ca(2+)](i) in 75% of astrocytes with an EC(50) of 12.7 +/- 3.1 nm. This rapid action of estradiol was blocked by the ER antagonist, ICI 182,780. The membrane-impermeable estradiol-BSA induced a [Ca(2+)](i) flux that was statistically similar to estradiol. Removal of extracellular Ca(2+) did not alter the effect of estradiol, but phospholipase C inhibitor U73122 (10 microm) and 2-aminoethoxydiphenyl borate (5 microm), an inhibitor of the inositol-1,4,5,-trisphosphate-gated intracellular Ca(2+) channel, significantly decreased the estradiol-induced [Ca(2+)](i) flux. Estradiol was unable to induce [Ca(2+)](i) flux in thapsigargin-depleted cells. These results indicate that estradiol mediates [Ca(2+)](i) flux in astrocytes through a membrane-associated ER that activates the phospholipase C pathway.