Facilitative urea transporters play a vital role in the production of concentrated urine in the mammalian kidney (1). The renal urea transporter UT-A3 is expressed on the basolateral membrane of mouse inner medullary collecting duct cells (2). Previous experiments using an MDCK cell line stably expressing mUT-A3 (MDCK-mUT-A3) have shown that the hormone vasopressin acutely regulates UT-A3-mediated basolateral urea transport (3). In this study, we have investigated the effect of vasopressin on the localization of UT-A3 transporters. Utilising serial centrifugation to separate protein samples from various sub-cellular fractions, we probed immunoblots of MDCK-mUT-A3 protein with the UT-A3 antibody ML446 (2). The addition of 10^-6M vasopressin for 60 minutes shifted the predominant 40-45 kDa UT-A3 signal from 200,000g samples (intracellular membranes) to 17,000g samples (plasma membranes). Vasopressin therefore significantly increased the UT-A3 plasma membrane signal (P<0.05, ANOVA, n = 3). In contrast, it had no such effect on a 100 kDa NaKATPase signal in the same samples (NS, ANOVA, n = 3), suggesting that it was a specific response. In addition, the presence of 10µM H89, a protein kinase A inhibitor, did not prevent the vasopressin-stimulated increase in UT-A3 plasma membrane signal (P<0.05, ANOVA, n = 3). In conclusion, this study suggests that vasopressin increases basolateral urea transport in MDCK-mUT-A3 cells at least partly by increasing localization of UT-A3 to the basolateral membrane.