The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.