PURPOSE We have previously provided molecular evidence that aquaporins (AQP) are expressed by human urothelium, suggesting a potential role in water and solute transport (Rubenwolf et al, 2008). The aim of this work was to investigate factors that regulate AQP expression in human urothelium and to develop functional assays to examine the potential role in urothelial physiology. MATERIAL AND METHODS Normal Human Urothelial (NHU) cell lines were established and the effect on AQP protein expression of exposing NHU cell cultures to osmotic stress was examined by immunochemistry. The barrier properties of differentiated urothelial constructs were assessed by measuring transepithelial electrical resistance (TER) and permeability coefficients for 3H water and 14C urea. HgCl2 was used as a non-competitive AQP channel blocker. RESULTS AQP 3 protein expression was shown to be up-regulated by up to 13-fold in response to changing the culture medium osmolality from 295 to 500 mosm/kg. Differentiated urothelium revealed a significant barrier function (average TER: 3680 Ω.cm 2); average diffusive water and urea permeability coefficients (Pd) were 10.4x10-5 and 3.7x10-5 cm/sec, respectively. Mean osmotic permeability coefficients (Posm) were 1.8-fold higher than corresponding diffusive permeability coefficients. A significant, dose-dependent (up to 4-fold) decrease of both water and urea flux across urothelium was observed upon application of HgCl2. CONCLUSIONS Our results demonstrate for the first time that expression of AQP 3 is regulated by osmolality, which suggests that urothelium is capable of reabsorbing water in response to the body's hydration status. The low, but finite, permeability of cultured urothelium to water and urea, which was reduced upon AQP inhibition, is also highly supportive of a previously unrecognised role for AQPs in body fluid regulation with potential implications on urinary tract physiology and urinary continence.