Abstract When male rats of certain strains are fed a diet with 3% or more Na saccharin, their urinary bladders develop epithelial hyperplasia and a greater incidence of tumors. Since the daily dose of saccharin is high, a link between tumor formation and the disruption of urothelial physiologic and biochemical processes has been sought. We fed male and female Sprague-Dawley rats a saccharin-free or 7.5% Na saccharin diet for 1 month. Excised bladders were mounted in flux chambers and exposed to Krebs-Ringer bicarbonate solution (KRB) or urine. Bioelectric properties and 22Na, 36Cl, and [ 14C]mannitol or [ 3H]mannitol unidirectional fluxes were measured by conventional techniques. No differences were noted between bladders from male and female animals or between Na saccharin-fed animals and animals fed the saccharin-free diet. When both surfaces of the epithelium were exposed to KRB, transepithelial dc conductance fell over 4 hr to 50% of the initial value. Conductance averaged 1.4 mS/cm 2. Transepithelial potential difference (PD) was usually lumen negative and averaged 0.7 mV. Unidirectional permeability coefficients for 36Cl, 22Na, and radiomannitol were symmetric, proportional to conductance, and followed a rank order compatible with unrestricted passive diffusion. Exposure of the bladder lumen to urine from animals fed saccharin-free or Na saccharin diet hyperpolarized the transepithelial PD by more than 5 mV and raised conductance nearly threefold. Permeability coefficients remained symmetric and compatible with passive diffusion. Exposure of the lumen to solutions with the K +, Na +, and Cl − concentrations and osmolality of urine simulated the conductance and PD effects of urine. We conclude that Na saccharin feeding or urine with saccharin does not uniquely affect the permeability of the excised preparation. Small hydrophilic solutes appear to cross the bladder epithelium through paracellular channels which increase in aggregate area during exposure of the lumen to urine. The hyperpolarization induced by lumenal urine is the consequence of the transepithelial K + gradient.