The complexity of estrogen actions mainly relies to the presence of different identified receptors (ERα, ERβ, their isoforms, and GPR30/GPER) and their discrete cellular distribution. Depending on the localization of the receptor that mediates estrogen effects, nuclear and extra-nuclear actions have been described. The latter can trigger a number of signaling events leading also to transcriptional modifications. In an attempt to clarify the nature of the receptor(s) involved in the membrane initiated effect of estrogens on gene expression, we performed a whole transcriptome analysis of breast cancer cell lines with different receptor profiles (T47D, MCF7, MDA-MB-231, SK-BR-3). A pharmacological approach was conducted with the use of estradiol (E(2)) or membrane-impermeable E(2)-BSA in the absence or presence of a specific ERα-β or GPR30/GPER antagonist. Our results clearly show that in addition to the ERα isoforms and/or GPR30/GPER that mainly mediate the transcriptional effect of E(2)-BSA, there is a specific transcriptional signature (found in T47D and MCF-7 cells) suggesting the presence of an unidentified membrane ER element (ERx). Analysis of its signature and phenotypic verification revealed that important cell function such as apoptosis, transcriptional regulation, and growth factor signaling are associated with ERx.