In this study, we report a reliable technique for the harvest, cultivation and expansion of monoculture of NMU. The NMU were harvested by two methods, directly from the urothelium in vivo and indirectly from the urothelial outgrowths of bladder explant cultures. Primary cultures and subsequent subcultures were propagated in the mixture of media MCDB 153 and Advanced-DMEM, and conditioned medium. Primary urothelial cells required an initial plating density of 1 x 10(5) viable cells/cm2 for survival, while passaged cells needed lower plating densities (1 x 10(4) viable cells per cm2). The cultured cells were identified as urothelial by their epithelioid morphology and by the positive immunofluorescence labelling of tight junctional proteins, occludin and ZO-1, adherens protein E-cadherin and cytoskeletal protein cytokeratin 7. Markers of highly differentiated urothelial cells, cytokeratin 20 and uroplakins, were not expressed. Furthermore, the immunofluorescence labelling of occludin and cytokeratin 7 was not detected in later passages when urothelial cells replicated at a high rate. In spite of the use of conditioned medium derived from V79 fibroblast cell culture supernatant, the NMU in the primary cultures and subsequent subcultures expressed a basal/intermediate cell phenotype. In conclusion, we demonstrate that homogeneous long-term culture of NMU can be developed. Since powerful transgenic tools exist to manipulate the mouse genome, our findings should help design the mouse in vitro systems for studying the control mechanisms of urothelial cell proliferation, stratification and differentiation in health and disease.