OBJECTIVE: The bladder urothelium is comprised of basal, intermediate, and apical urothelial cells. Ability to functionally study an individual urothelial cell while preserving its in situ location would represent an advance in bladder urothelial biology. MATERIALS AND METHODS: Mice were euthanized and cardiac perfused with phosphate-buffered saline. Bladders were then excised. Urothelial sheets were dissected off the lamina propria using forceps with a microscope (5 × magnification). Sheets were stained with H&E. In separate experiments, cell-attached electrophysiology was performed by placing urothelial sheets in Ringer’s bath solution, with either basal or apical surface down. Using 40 × magnification, individual urothelial cells from different layers were identified. Potassium currents on these cells were measured in situ using single channel patch-clamp technique. RESULTS: Histology revealed that urothelial sheets were free of lamina propria and smooth muscles. The proportion of apical cells, compared to proportion of intermediate and basal cells, with measurable potassium currents was considerably higher in apical cells (69% of apical versus 16% of intermediate and 18% of basal cells). Of the active patches detected in apical cells, 100% of these patches showed a 43 pS current conductance. For intermediate and basal cells, 75-83% demonstrated a 43 pS current and 17-25% demonstrated a 22 pS current. Single cells, from all 3 layers, could also be individually microdissected completely off the urothelium. CONCLUSIONS: A novel approach was developed in which individual urothelial cells within the multi-layered urothelium were identified and functionally studied in situ. Electrophysiologic characterization revealed different phenotype between the cells from different layers. Single cells from an identified layer can be harvested off the urothelium allowing for other studies. This technique allows investigators to study various cellular functions while preserving cellular location within the urothelium.