The efficient handling of urea by the kidney is crucial for the development of maximally concentrated urine. The inner medullary collecting duct (IMCD) plays an important role in urea reabsorption. The transport of urea across the IMCD is governed by the UT-A family of urea transporters, with UT-A3 providing a pathway across the basolateral membrane. In the current study we have investigated the role of the N-terminal of UT-A3 on membrane targeting. All clones were N-terminally tagged with eGFP. N-terminal truncations were created by standard PCR protocols, verified by sequencing and subcloned into the eGFP expression plasmid pEGFP-C2. The 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 times for each construct. When expressed in MDCK II cells, mUT-A3 is delivered to the basolateral membrane of the cell (Stewart et al., 2004). We have shown previously (Cooper and Collins, 2006) that removing the initial 55 residues of UT-A3 (M55-start) does not affect urea-transporting ability. However when expressed in MDCK II cells, M55-start targets to the apical membrane. When we removed the initial 25 residues (S25-start) we found that targeting was once again basolateral. When expressed in LLC-PK1 cells mUT-A3 targeted to the apical membrane. LLC-PK1 cells lack the μ1B subunit of the adaptor protein 1 complex (AP1-μ1B). The μ1B subunit has been implicated in basolateral targeting of a number of proteins (Folsch et al., 1999). We expressed mUT-A3 and M-55start in LLC-PK1 cells stably transfected with AP1-μ1B. Although mUT-A3 was now targeted to the basolateral membrane, the M55-start clone was still resident in the apical membrane. Our results indicate that a region between residues 25 and 55 in the N-terminal is involved in the basolateral targeting of mUT-A3, probably via an interaction with the AP1-μ1B subunit.