Urea transport in the kidney plays an important role in urinary concentration and nitrogen balance. Recently, three types of urea transporters have been cloned, UT1 and UT2 from rat and rabbit kidney and HUT11 from human bone marrow. To elucidate the physiological role of the latter urea transporter, we have isolated the rat homologue (UT3) of HUT11 and studied its distribution of expression and functional characteristics. UT3 cDNA encodes a 384 amino acid residue protein, which has 80% identity to the human HUT11 and 62% identity to rat UT2. Functional expression in Xenopus oocytes induced a large (approximately 50-fold) increase in the uptake of urea compared with water-injected oocytes. The uptake was inhibited by phloretin (0.75 mM) and pCMBS (0.5 mM) (55 and 32% inhibition, respectively). Northern analysis gave a single band of 3.8 kb in kidney inner and outer medulla, testis, brain, bone marrow, spleen, thymus, and lung. In situ hybridization of rat kidney revealed that UT3 mRNA is expressed in the inner stripe of the outer medulla, inner medulla, the papillary surface epithelium, and the transitional urinary epithelium of urinary tracts. Co-staining experiments using antibody against von Willebrand factor showed that UT3 mRNA in the inner stripe of the outer medulla is expressed in descending vasa recta. These data suggest that UT3 in kidney is involved in counter current exchange between ascending and descending vasa recta, to enhance the cortico-papillary osmolality gradient. In situ hybridization of testis revealed that UT3 is located in Sertoli cells of seminiferous tubules. The signal was only detected in Sertoli cells associated with the early stages of spermatocyte development, suggesting that urea may play a role in spermatogenesis.