Facilitative UT-B urea transporters play a crucial role in the human bladder (1), protecting urothelial cells from the damaging effects of urea (2). Allelic variation in these UT-B transporters has been linked to the risk of developing bladder cancer (3), so it is imperative to understand the physiological regulation of the UT-B proteins. Our previous studies utilised the RT4 cell line model of human urothelial cells to show that NaCl-induced, but not mannitol-induced, increases in external osmolality upregulated the abundance of UT-B transporter protein (4). In this current study, we further investigated the effects of urea-induced changes in external osmolality using the same cell line. RT4 cells were cultured for 48 hours in media with a range of additional urea concentrations and then membrane-enriched protein samples were prepared from treated flasks of cells. Next, these samples were used for semi-quantitative western blotting experiments and then densitometry analysis was performed (with data given as mean values ± SEM). Compared to control experiments (50 ± 7, N=4), no significant change in UT-B protein abundance occurred with 10mM urea treatment (56 ± 6, N=4, NS, ANOVA), but significant increases did occur with both 50mM (67 ± 4, N=4, P<0.05, ANOVA) and 100mM urea (66 ± 1, N=4, P<0.05, ANOVA) treatments. Importantly, while there was significant specific increase in glycosylated UT-B protein with 50mM urea treatment (86 ± 5 versus 59 ± 8, P<0.05, N=4, ANOVA), there was no change in NaKATPase protein (27 ± 4 versus 32 ± 6, NS, N=4, ANOVA). Overall, these data show that urea-induced changes in external osmolality specifically upregulated levels of glycosylated UT-B urea transporter in the RT4 cell line. As glycosylation is crucial for UT-B function, we suggest that the urea-induced changes observed may be of important physiological significance.