Like other steroid hormone receptors, estrogen receptor-alpha (ERalpha) is a substrate for protein kinases, and phosphorylation has profound effects on the function and activity of this receptor. A number of different kinases have been implicated in ERalpha regulation. In this report we show by mutational analysis and in vitro kinase assays that ERalpha is a substrate for glycogen synthase kinase-3 (GSK-3) in vitro and is phosphorylated on two sites, the Ser-102, -104, and -106 motif and Ser-118, both located in the N-terminal transcription activation function (AF-1) domain. GSK-3 forms a complex with ERalpha in vivo as demonstrated by co-immunoprecipitation from cell lysates. The GSK-3 inhibitor lithium chloride was used to determine the role of GSK-3 in phosphorylation of Ser-102, -104, and -106 and Ser-118 in vivo and to explore the role of these serines in the regulation of ERalpha function. Treatment of cells with lithium chloride resulted in dephosphorylation of Ser-104 and -106 and Ser-118, which suggests these serine residues as targets for GSK-3 in vivo. Our results further suggest that ERalpha phosphorylation by GSK-3 stabilizes ERalpha under resting conditions and modulates ERalpha transcriptional activity upon ligand binding. Inhibition and constitutive activation of GSK-3, both, resulted in inhibition of ERalpha transcriptional activity, indicating a function of active as well as inactive GSK-3 in ERalpha regulation. These findings uncover a novel mechanism for the regulation of ERalpha-mediated estrogen signaling controlled by a dual action of GSK-3.