Urea is the largest solute component of urine and efficient urea handling is crucial in the generation of maximally concentrated urine. Urea movement across the nephron is facilitated by the UT-A urea transporters. Previously we have shown that UT-A3 is expressed on the basolateral membrane of the inner medullary collecting duct (Stewart et al, 2004) and that 7 amino acids between residues 450–456 in the C-terminus are important for urea transport (Collins & Cooper, 2006). The current study further investigates the role of this 7aa region. All clones were N-terminally tagged with eGFP. Mutations were introduced using quikchange and confirmed by sequencing. Oocytes were isolated from Xenopus laevis, injected with 1.5ng of the appropriate cRNA or 50nl of water. Urea transport was assessed by a 90s uptake of 14C-labelled urea, 3-4 days post injection (Fenton et al, 2000). Each group contained at least 10 oocytes from 2 or more animals. Statistical analysis was by one way ANOVA coupled with Student-Newman-Keuls test and significance was assumed at the 5% level. MDCK II cells were grown on permeable filters and transiently transfected with 1µg of cDNA. Cells were fixed and the localisation of each construct determined by immunofluorescence and confocal microscopy. The apical membrane was labelled with TRITC-conjugated peanut agglutinin and the basolateral membrane was labelled using a primary antibody against β-catenin and a Cy-5 conjugated secondary antibody. Transfections were replicated 3–5 times for each construct. The C-terminus sequence of mUT-A3 is 449VRRSEEEKSPNGD460. As found previously, urea uptake was observed for the K456X mutant but not the V449X mutant. A stop codon was inserted at 453, and the E453X mutant also retained the ability to transport urea. When transiently expressed in polarised MDCK II cells, mUT-A3, E453X and 456X co-localised with β-catenin in the basolateral membrane. In contrast, in cells transfected with V449X, fluorescence was exclusively intracellular. These results suggest that the C terminal of mUT-A3 is important in trafficking. The V449-K456 region reveals a high density of negatively charged residues and a putative PKA phosphorylation site (450RRS452). Starting with the E453X mutant we mimicked phosphorylated/dephosphorylated states at S452. With a charged residue at 452 (S452D-E453X) urea uptake was intact, but if the charge was removed (S452A-E453X), urea transport was lost. Returning to the full length clone, mutating the region of negative charge (452SEEE455) to 452AAAA455 or 453AAA455 did not interfere with urea uptake or trafficking. The last residue of mUT-A3 is negatively charged. Our results suggest that the presence of a negative charge at the end of the protein rather than the composition of the region V449-K456 is important for delivery to the plasma membrane.