The Nuclear Factor (NF)-κB family of transcription factors controls expression of genes which promote cell growth, survival, and neoplastic transformation. Recently we demonstrated aberrant constitutive activation of NF-κB in primary human and rat breast cancer specimens and in cell lines. Overexpression of the epidermal growth factor receptor (EGFR) family member Her-2/neu, seen in approximately 30% of breast cancers, is associated with poor prognosis. Previously, Her-2/neu has been shown to signal via a phosphatidylinositol 3 (PI3)-kinase to Akt/protein kinase B (PKB) pathway. Since this signaling pathway was recently shown to activate NF-κB, here we have tested the hypothesis that Her-2/neu can activate NF-κB in breast cancer. Overexpression of Her-2/neu and EGFR-4 in Ba/F3 cells led to constitutive PI3-and Akt kinase activities, and induction of classical NF-κB (p50/p65). Similarly, a tumor cell line and tumors derived from MMTV-Her-2/neu transgenic mice displayed elevated levels of classical NF-κB. Engagement of Her-2/neu receptor downregulated the level of NF-κB. NF-κB binding and activity in the cultured cells was reduced upon inhibition of the PI3-to Akt kinase signaling pathway via ectopic expression of kinase inactive mutants, incubation with wortmannin, or expression of the tumor suppressor phosphatase PTEN. Inhibitors of calpain, but not the proteasome, blocked IκB-α degradation. Inhibition of Akt did not affect IKK activity. These results indicate that Her-2/neu activates NF-κB via a PI3-to Akt kinase signaling pathway that can be inhibited via the tumor suppressor PTEN, and is mediated by calpain rather than the IκB kinase complex.