Estrogens regulate multiple activities in breast cancer cells, including proliferation. Whereas these hormones are most commonly known to regulate gene transcription through direct interaction with estrogen receptors (ERs) and with specific DNA sequences of target genes, recent studies show that ER also activates a number of rapid signaling events that are initiated at the cell membrane. To study the membrane-initiated effects of estrogen and separate them from the activities initiated by the nuclear localized ER in human breast cancer cells, we generated MDA-MB-231 breast cancer cell lines that have stably integrated either the wild-type nuclear form of ER (WT-ER) or a modified, membrane-targeted ER (MT-ER) that lacks a nuclear localization sequence and is dually acylated with a myristoylation sequence at the N terminus and a palmitoylation sequence at the C terminus. We demonstrate that MT-ER is membrane localized in the absence of estradiol (E2), showing punctate membrane and cytoplasmic speckles after E2 exposure. In contrast to WT-ER, MT-ER was not down-regulated by E2 or by antiestrogen ICI 182,780 exposure, and MT-ER failed to regulate endogenous E2-responsive genes highly up-regulated by WT-ER. Cells expressing MT-ER showed a greater serum response element-mediated transcriptional response that was partially inhibited by antiestrogen ICI 182,780. The MT-ER and WT-ER differentially altered ERK1/2 and Akt activities and the proliferation of breast cancer cells in response to E2. Hence, this study reveals distinct actions of the MT-ER vs. the WT-ER in effecting estrogen actions in breast cancer cells.