Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism (Fenton et al. 2004). UT-A3 is a phloretin-sensitive urea transporter expressed on the basolateral membrane of inner medullary collecting duct cells (Stewart et al. 2004). In this study, we have produced a MDCK cell line that stably expresses myc-tagged UT-A3, and investigated the resulting urea transport in these MDCK:UT-A3 cells. Radioactive ¹⁴C-labelled urea flux experiments showed that during basal conditions there was no difference in basolateral urea uptake into control MDCK cells (1.72 ± 0.22 nmol cm^-2 min^-1, n=16) and MDCK:UT-A3 cells (1.99 ± 0.24 nmol cm^-2 min^-1, n=16) (NS, unpaired t test). However, while pre-incubation for 60 min in 10^-6 M arginine vasopressin (AVP) had no effect on urea uptake into control MDCK cells (1.94 ± 0.09 nmol cm^-2 min^-1, n=4, NS, ANOVA), it significantly stimulated urea uptake into MDCK:UT-A3 cells (6.12 ± 0.68 nmol cm^-2 min^-1, n=4, P<0.05, ANOVA); as did 60 min pre-incubation with 10^-6 M AVP (5.23 ± 0.66 nmol cm^-2 min^-1, n=4, P<0.05, ANOVA). Further investigation showed that this 10^-6 M AVP response was in fact biphasic, with an initial peak after 10 min (5.31 ± 0.70 nmol cm^-2 min^-1, n=4, P<0.05, ANOVA) followed by a larger response after 60 min (7.07 ± 0.74 nmol cm^-2 min^-1, n=4, P<0.05, ANOVA). Importantly, the 60 min AVP response was significantly inhibited by 500 μM phloretin (73 ± 8% of control, n=4, P<0.05, ANOVA). Finally, MDCK:UT-A3 urea uptake was also stimulated by 10 μM forskolin (5.53 ± 0.78 nmol cm^-2 min^-1, n=4, P<0.01, ANOVA), 250 μM 8-bromo-cAMP (3.74 ± 0.23 nmol cm^-2 min^-1, n=4, P<0.05, ANOVA) or 1 mM ATP (4.73 ± 0.56 nmol cm^-2 min^-1, n=4, P<0.05, ANOVA). In conclusion, our results indicate that phloretin-sensitive UT-A3 urea transport can be regulated by AVP, possibly via intracellular increases of cAMP and/or calcium.