Urea transporters play an integral role in the urinary concentration process. UT-A3 is expressed basolaterally in cells lining the inner medullary collecting duct (Stewart et al. 2004). Although much is known about the regulation and distribution of UT-A3, we have little idea how these proteins function at the molecular level. The current study evaluates the role of the N- and C-termini in the function of the mouse urea transporter UT-A3 (mUT-A3). In all experiments mUT-A3 and its mutations have been N-terminally tagged with eGFP. Truncation and point mutations were constructed using standard PCR techniques. All mutations were confirmed by sequencing. Oocytes were isolated from Xenopus laevis and injected with 1.5ng of cRNA encoding mUT-A3, its mutants or 50nl of water. Urea transport was assessed 3-4 days after injection by measuring uptake of ¹⁴C-labelled urea as described previously (Fenton et al. 2000). Statistical analysis was performed using one way ANOVA coupled with the Student-Newman-Keuls test. Results were obtained from at least 10 oocytes, isolated from 2 or more animals. Significance has been assumed at the 5% level. The N-terminal of mUT-A3 was shortened, to start at residues M55 and M111. The M55-start mutant was functional when expressed in oocytes. M111-start, was non-functional suggesting a region between M55 and M111 is important for mUT-A3 function. A second series of truncated mutants were constructed with the N-terminal starting at S68, G94 and A103. All of these mutants were functional. In full length mUT-A3, deleting residues 103 to 113 (mUT-A3-Δ103-113) prevented urea uptake. When expressed in non-polarised MDCK cells, mUT-A3-Δ103-113 was trafficked to the plasma membrane and demonstrated a distribution pattern similar to wild-type mUT-A3. Taken together these results suggest that the 8-aa region between residues 103 and 113 is required for normal transport function but is not linked to protein trafficking. To investigate the role of the C-terminus, a series of truncation mutants were constructed. mUT-A3 has a predicted open reading frame of 460 amino acids. We inserted stop codons at residues 285, 315, 409, 429, 449 and 456. Urea uptake was not observed in oocytes expressing the E285X, I315X, W409X, Y429X V449X mutants. However, deleting the final 4 amino acids (K456X) did not reduce urea uptake compared to full length mUT-A3. These results suggests the 7aa between residues 450–456 are important for urea transport, although the precise role of this region in mUT-A3 function still needs to clarified.