Normal human uroepithelial cells (HUC) were transformed with simian virus 40 (SV40) in vitro. SV40-transformed HUC (SV-HUC) were selected by their ability to survive senescence which normally occurs in HUC between passages 4 and 6. At passage 6, 100% of SV-HUC stained positive for SV40 T-antigen. The epithelial nature of SV-HUC was confirmed by positive staining for human cytoplasmic keratins in all cells. SV-HUC have altered growth characteristics compared to HUC including the capacity to grow on plastic, independent of a collagen-gel substrate; loss of the dependence on medium supplements for optimal growth, loss of the dependence on feeder cells for growth at clonal density, and an apparently unlimited lifespan in culture (greater than 2 years). Although SV-HUC have an increased percentage of viable cells and increased saturation density compared to HUC, the generation time of SV-HUC during log phase is similar to that of HUC. Cultures of SV-HUC are epithelial in appearance and show some morphological heterogeneity in cell size and shape. At the ultrastructural level, SV-HUC have numerous alterations such as, irregularly shaped nuclei and nucleoli, pleomorphic microvilli, and the lack of a glycocalyx on the cell surface. In addition, SV-HUC does not stratify in culture, suggesting an inability to differentiate. Unlike HUC, SV-HUC are capable of growth in soft agarose, a property which increased with serial passage. Yet, through at least P50, SV-HUC remained nontumorigenic as determined by the inability to form tumors in athymic nude mice. This cell line of human epithelial origin may be suitable for studying the conversion of cells to tumorigenicity by subsequent treatment with another oncogenic agent.