17Beta-estradiol (E2) induces proliferation and c-fos gene expression in MCF-7 cells and both responses are partially blocked by wortmannin and LY294002 which are inhibitors of phosphatidylinositol-3-kinase (PI3-K). Analysis of the c-fos gene promoter shows that the effects of wortmannin and LY294002 are associated with inhibition of E2-induced activation through the serum response factor (SRF) motif within the proximal serum response element at -325 and -296. E2 activates constructs containing multiple copies of the SRF (pSRF) and a GAL4-SRF fusion protein; these responses are accompanied by PI3-K-dependent phosphorylation of Akt and inhibited by wortmannin/LY294002, the antiestrogen ICI 182780, but not by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD98059. Using a series of kinase inhibitors and dominant negative kinase expression plasmids, it was shown that the non-genomic activation of SRF by E2 was associated with src-ras-PI3-K pathway, thus, demonstrating hormonal activation of the SRE through src-ras activation of both PI3-K- and MAPK-dependent signaling pathways.