Long-term estrogen deprivation causes hypersensitivity of MCF-7 cells to the mitogenic effect of estradiol (E2) which is associated with activation of mitogen-activated protein kinase (MAPK). However, several lines of evidence indicate that MAPK activation is not the exclusive mechanism for E2 hypersensitivity and multiple signal pathways might be involved. The current study explores the possible role of the PI3 kinase (PI3K) pathway in development of E2 hypersensitivity. Basal PI3K activity in long-term estrogen deprived MCF-7 cells (LTED) was elevated as evidenced by increased phosphorylation of three downstream effectors, Akt, p70 S6 kinase, and eukaryotic initiation factor-4E binding protein (4E-BP1), which was blocked by the specific inhibitor of PI3K, LY294002. Dual blockade of both MAPK and PI3K completely reversed E2 hypersensitivity of LTED cells. Enhancement in aromatase activity is another phenomenon accompanied with E2 hypersensitivity. In aromatase over-expressing MCF-7 cells, aromatase activity was reduced by inhibitors of MAPK and PI3K suggesting the involvement of protein phosphorylation in the regulation of aromatase activity. Our data suggest that in addition to the MAP kinase pathway, activation of the PI3 kinase pathway is involved in E2 hypersensitivity, which develops during adaptation of MCF-7 cells to the low estrogen environment.