CD40, a tumour necrosis factor (TNF) receptor family member, is expressed in a variety of cell types, including B lymphocytes, macrophages, fibroblasts, endothelial and epithelial cells, and this widespread expression is likely to account for its central role in normal physiology and disease pathogenesis. In this study, we provide evidence to support a role for constitutive CD40 signalling in cell transformation. We show that the ligand for CD40 (CD40L/CD154) is expressed in CD40-positive human breast tumour biopsies, suggesting that the constitutive activation of the CD40 receptor in vivo may contribute to the oncogenic process. Coexpression of CD40 and CD40L confers oncogenic effects on immortalized human epithelial cells in vitro, increasing their proliferation, motility and invasion. Expression of LMP:CD40, a hybrid molecule comprising the N-terminus and transmembrane domains of the Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) fused to the cytoplasmic tail of CD40, mimics a constitutively active CD40 receptor and promotes the transformation of immortalized rodent fibroblasts in vitro and their oncogenicity in vivo. The observed effects of aberrant CD40 activation on cell transformation are largely diminished upon suppression of the oncogenic NF-kappaB signalling pathway. Taken together, our results suggest a role for the constitutive engagement of the CD40L/CD40/NF-kappaB activation pathway in cell transformation and neoplastic growth. Strategies that neutralize this pathway may therefore be useful in cancer treatment and prevention.