NFB plays an important role in mammary gland development and breast cancer. We previously demonstrated that TNF stimulates growth of mammary epithelial cells (MEC) in a physiologically relevant 3D primary culture system, accompanied by enhanced DNA-binding of the NFB p50 homodimer. To further understand the mechanism of TNF-stimulated growth of primary MEC, the requirement for NFB1/p50 and the role of cyclin D1 in TNF-stimulated growth were examined. TNF induced the formation of DNA-binding complexes of p50 and p52 with their coactivator bcl3 in MEC nuclear extracts. Concomitantly, TNF increased the binding of NFB proteins to the B site on the cyclin D1 promoter, and increased expression of cyclin D1 mRNA and protein. Using MEC from p50 null mice, we found that p50 was not required for TNF-induced growth nor for up-regulation of cyclin D1. However, TNF induced a p52/RelB NFB DNA-binding complex in p50 null MEC nuclear extracts. In addition, we found that in wild-type MEC, TNF stimulated the occupancy of p52 and RelB on the cyclin D1 promoter B site, while p50 was present constitutively. These data suggest that in wild-type MEC, TNF stimulates the interaction of bcl3 with p50 and p52, and the binding of p52, as well as RelB, to cyclin D1 promoter B sites, and as a consequence stimulates the growth of MEC. In the absence of p50, p52 and RelB can compensate for p50 in TNF-stimulated growth and cyclin D1 induction in MEC.