The aberrant expression of the nuclear factor-kappaB (NF-kappaB) c-Rel subunit that occurs in many human breast cancers can play a causal role in tumorigenesis as judged by findings with a mouse mammary tumor virus (MMTV)-c-rel transgenic mouse model, in which 31.6% of mice developed one or more mammary tumors after a long latency. Interestingly, none of the cell lines established from the mammary tumors grew in soft agar. To begin to test the hypothesis that a prototypic carcinogen insult can promote a more invasive, mesenchymal phenotype, a cell line established from a MMTV-c-rel mammary tumor rel-3983 was treated in culture with the polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA; rel-3983D cells) or DMSO vehicle (rel-3983V cells). Rel-3983D cells displayed an increased rate of proliferation, displayed growth to a higher cell density, and acquired the ability to grow in soft agar and in Matrigel compared with the parental rel-3983 or vehicle-treated rel-3983V cells. Consistent with a more mesenchymal phenotype, rel-3983D cells showed loss of E-cadherin expression as judged by immunofluorescence microscopy. Compared with control cells, rel-3983D displayed increased NF-kappaB binding and higher levels of the NF-kappaB transactivating subunits c-Rel, RelA, and RelB, which seemed functional as judged by induction of c-Myc and vimentin, products of two NF-kappaB target genes. Ectopic expression of a super repressor mutant of IkappaB-alpha reduced rel-3983D cell growth and invasive morphology in Matrigel, confirming the role of NF-kappaB in epithelial to mesenchymal transition (EMT). Thus, DMBA treatment of c-Rel-transformed mammary tumor cells in culture is shown here for the first time to result in EMT via activation of NF-kappaB. The aberrant c-Rel expression present in most human breast cancers suggests that this mechanism may play an important role in carcinogenesis.