The renal UT-A urea transporters UT-A1, UT-A2 and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In this study we have investigated the effect of ubiquitination on UT-A plasma membrane expression in MDCK cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal trans-epithelial urea flux across MDCK-rUT-A1 and MDCK-mUT-A2 monolayers (P<0.01, n = 3, ANOVA), and also increased DMU-sensitive, AVP-stimulated urea flux (P<0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P<0.01, n = 4, ANOVA) in a concentration dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P<0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P<0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily utilised by vasopressin to produce its physiological effects.