Background: The tumor necrosis factor (TNF) superfamily of ligands and receptors mediates immune cell survival. Some members possess a death domain, a protein motif that functions to transmit apoptotic signals, whereas others, such as CD40, do not. CD40 is expressed by both normal and malignant epithelial cells. To investigate the functional significance of this expression, we studied the effects of ligation of CD40, Fas, and TNF receptors (TNFRs) on the proliferation and survival of normal and malignant human urothelial cells and urothelial cells with disabled p53 function. Methods: Normal and malignant human urothelial cells were cultured with soluble TNF family agonists (CD40 ligand [CD40L], TNF-α, anti-Fas antibody, or cocultured with mouse fibroblasts stably transfected with plasmids that caused the cells to constitutively express CD40L or CD32; cell proliferation was estimated by an [3H]thymidine incorporation assay, and apoptosis was determined by Annexin V staining and by a DNA fragmentation assay. Messenger RNA levels for CD40 and potential downstream effector molecules were quantified by polymerase chain reaction-based and ribonuclease protection assays, respectively, and nuclear factor (NF) κB nuclear translocation was detected by immunofluorescence. All statistical tests were two-sided. Results: Soluble trimeric CD40L inhibited the growth of normal and malignant urothelial cells but did not induce apoptosis. Cell surface-presented CD40L induced massive apoptosis in CD40-positive transitional cell carcinoma cells but not in normal urothelial cells. Normal cells underwent CD40L-mediated apoptosis only in the presence of other TNFR agonists. An agonistic anti-CD40 antibody presented on the surface of CD32-transfected fibroblasts also induced apoptosis in transitional cell carcinoma cells and in normal urothelial cells. Apoptotic responses of tumor (but not normal) cells to soluble agonists were enhanced by blocking protein synthesis. Karyotypically normal urothelial cells with disabled p53 function underwent apoptosis during coculture with CD40L-expressing fibroblasts alone but were not additionally sensitive to additional TNFR agonists. Conclusions: Susceptibility to CD40 ligation-induced apoptosis may be a novel mechanism for eliminating neoplastically transformed urothelial cells. Loss of CD40 expression may be an important adaptive mechanism for transitional cell carcinoma development and progression.