In addition to 17-estradiol binding, estrogen receptor (ER) transcriptional activity could be controlled by intracellular kinase signaling pathways activated by growth factors. Here we present evidence suggesting that glycogen synthase kinase 3 (GSK3), an effector kinase of the phosphatidyl inositol 3 kinase (PI3K) pathway, may affect ER activity in N2a neuroblastoma cells. LiCl, sodium valproate and SB415286, three inhibitors of GSK3, dose dependently blocked ER-mediated transcription. In contrast, overexpression of wild-type GSK3, but not of a mutant inactive form, increased ER-dependent gene expression. Pharmacological or genetic inhibition of the PI3K/Akt pathway, whose activity is inversely correlated with that of GSK3, increased ER-mediated transcription and this effect was blocked by GSK3 inhibitors. As in other cell types, insulin-like growth factor I (IGF-I) increased ER activity in absence of estradiol by a mechanism independent of PI3K. In contrast IGF-I decreased ER activity in the presence of estradiol and this effect was mediated by PI3K. We further observed a regulated interaction between -catenin, one of the main GSK3 nuclear targets, and ER. Transfection with a non-degradable mutant of -catenin blocked the increase in ER transcriptional activity induced by the PI3K inhibitor wortmannin, suggesting a role for -catenin in estrogen signaling. In addition, we investigated the regulation of ER protein levels as a potential mechanism for its regulation by the PI3K/GSK3 pathway: GSK3 blockade increased ER protein stability while PI3K inhibition decreased it. In summary, our findings suggest that ER-dependent gene expression in N2a cells is controlled by the PI3K/Akt/GSK3 signaling pathway.