It has been a common belief that estrogen regulates cellular responses through binding to its receptor, the estrogen receptor (ER). In the nucleus, estrogen modulates the expression of estrogen-responsive genes through the action of the ER at the transcriptional level. In the cytoplasm, the ER-dependent signaling pathway has been shown to be involved in the activation of Akt and the downstream molecules. It is not clear, however, whether estrogen can modulate cytoplasmic signaling in an ER-independent manner. Human breast cancer cell lines without detectable ERs such as MDA-MB-435 and MDA-MB-231 were treated in estrogen-depleted medium followed by a brief treatment with estrogen. The activation of Akt was evaluated by a phosphoserine antibody. Our results showed that estrogen stimulated Akt activation, as indicated by phosphorylation at Ser(473) of the oncoprotein, in ER-negative breast cancer cells. Activation of Akt by estrogen in these cells was time and dose dependent and could be blocked by inhibitors of phosphatidylinositol 3'-kinase and Src kinase but not by estrogen antagonists. Our results provide evidence as well as the mechanism of the receptor-independent function of estrogen, in which the antiapoptotic factor Akt is activated.