The process of urea nitrogen salvaging (UNS) is vital for maintaining nitrogen balance in ruminant animals, such as cattle and sheep (Lapierre & Lobley 2001). An important step in UNS is the passage of urea across the rumen epithelia. Although the pathway is not yet clearly understood, the involvement of facilitative urea transporters has previously been indicated (Marini & Van Amburgh 2003). In this study, we characterize a bovine UT-B urea transporter ortholog and investigate the role of UT-B in the bovine rumen. We obtained a 2234bp bovine UT-B cDNA clone, BE665260, from BACPAC Resources (CHORI, USA). The identified open reading frame (75-1229bp) encoded a 384 amino acid protein, bUT-B, which shared 79% homology with human UT-B (GenBank accession number Q13336). When expressed in Xenopus oocytes, bUT-B produced a 4-fold increase in urea transport (P<0.001, ANOVA) that was significantly inhibited by 500mM phloretin (P<0.01, ANOVA) or 2mM thionicotinamide (P<0.01, ANOVA). Rumen tissue was obtained from humanely killed cattle (Bos taurus). Northern analysis detected a 3.8 kb UT-B transcript in bovine rumen, the presence of which was further confirmed by RT-PCR analysis. Immunoblotting studies of bovine rumen tissue, using a mouse UT-B antibody (Stewart et al. 2004), detected a 45-50kDa UT-B protein. Using 10μm sections of methanol fixed rumen epithelium, UT-B was immunolocalised to the plasma membrane of cells of the stratum basale, stratum spinosum and stratum granulosum; the stratum corneum was negative. Functional studies of trans-epithelial transport across rumen epithelium showed that bi-directional fluxes of ¹⁴C-labelled urea were similar (J_{lumen to blood} 19.5 ± 2.5 nmol.cm^-2.hr^-1 (SEM), n=15 measurements from 4 animals, J_{blood to lumen} 18.7 ± 2.5 nmol.cm^-2.hr^-1(SEM), n=15) and exceeded simultaneously measured ³H-mannitol fluxes by 2.5 fold and 1.97 fold respectively. Phloretin (1mM) inhibited a component of the trans-epithelial urea flux, suggesting that additional pathways to UT-B (both mediated and passive) are likely to exist. We therefore conclude that UT-B is present in the bovine rumen and is likely to participate in trans-epithelial urea transport. As such, bovine UT-B may play a role in the UNS process.