AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor-like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser(473) phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERalpha levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERalpha-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 micromol/L LY294002 or 10 micromol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.