Despite significant advances in treatment, breast cancer is still the second leading cause of cancer-related deaths in women in the United States. Therefore, significant efforts are being given to develop novel strategies for the prevention of breast cancer in recent years. Our laboratory and others have been studying the effects of a potential chemopreventive agent, indole-3-carbinol (I3C), in breast cancer cells. We have previously shown that I3C induces apoptosis in breast cancer cells and found that the induction of apoptotic processes was partly mediated by dysregulation of anti- and pro-apoptotic molecules. However, the precise molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells has not been fully elucidated. For the present study, we focused our investigation on important cell signaling molecules such as Akt and NF-kappaB during I3C-induced apoptosis in breast cancer cells. We found that I3C induces apoptotic processes in MCF10A-derived cell lines with premalignant (DCIS.com) and malignant (MCF10CA1a) phenotypes but not in nontumorigenic parental MCF10A cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that I3C specifically inhibits Akt kinase activity and abrogates the EGF-induced activation of Akt in breast cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA and NF-kappaB-Luc reporter constructs revealed that Akt gene transfection directly activates NF-kappaB, and this activation was completely abrogated by I3C treatment. In addition, I3C also abrogated the EGF-induced activation of NF-kappaB, which was mediated via the Akt signaling pathway. From these results, we conclude that there is a direct cross-talk between Akt and NF-kappaB pathways and that the inactivation of Akt and NF-kappaB activity plays important roles in mediating I3C-induced apoptosis in breast cancer cells. These results also suggest that I3C may be a potential chemopreventive agent by virtue of its selective apoptosis-inducing ability in premalignant and malignant breast epithelial cells.