Nuclear factor-κB (NF-κB) is usually maintained in an inactive form in the cytoplasm through its association with inhibitor of kappaB (IκB) proteins, and is activated upon stimulation of cells with a variety of signals. However, constitutive activation of NF-κB is observed in a number of cancers including breast cancer. The signaling pathways that are involved in constitutive NF-κB activation remain largely unknown. Using breast cancer cell lines derived from transgenic mice that overexpress specific oncogene/growth factors in the mammary gland, we show that heregulin but not her2/neu, c-Myc or v-Ha-ras plays a major role in constitutive NF-κB activation. Her2/neu potentiated tumor necrosis factor alpha (TNFα)-inducible NF-κB activation whereas c-Myc potentiated 12-o-tetracecanyolphorbol-13-acetate (TPA)-induced NF-κB activation. Heregulin-mediated NF-κB activation correlated with phosphorylation of epidermal growth factor receptor (EGFR) and ErbB3 but not her2/neu. Tryphostin AG1517, which inhibits heregulin-mediated phosphorylation of EGFR, her2/neu and ErbB3 reduced NF-κB activation. In contrast, emodin, which blocks phosphorylation of her2/neu by heregulin, failed to reduce NF-κB activation. These results suggest that heregulin induces NF-κB independent of her2/neu. PI3 kinase/AKT, protein kinase A (PKA) and IκB kinase appear to be downstream signaling molecules involved in NF-κB activation as specific inhibitors of these kinases but not inhibitors of ERK/MAP kinase or protein kinase C reduced heregulin-mediated NF-κB activation. Based on these results, we propose that heregulin increases the expression of pro-invasive, pro-metastatic and anti-apoptotic genes in cancer cells through autocrine activation of NF-κB, which leads to invasive and drug-resistant growth of breast cancer.