Urea transporters (UTs) effect rapid flux of urea across biological membranes. In the mammalian kidney, UT activity is essential for effective urine concentration. In bacteria, UT-mediated urea uptake permits intracellular urease to degrade urea to ammonia and CO2, a process which either buffers acid loads or provides nutrient nitrogen. In many bacterial species, uptake of urea and its metabolism play a critical role in pathogenesis. Although UT’s have been identified and cloned from mammals and bacteria, their function has not been well characterized, limiting our ability to determine, when structural information becomes available, the detailed mechanism of UT urea transport. We have characterized the urea transport channel protein ApUT from Actinobacillus pleuropneumoniae. Expression of ApUT in Xenopus oocytes induced a 3 fold increase in 14C urea uptake compared to water injected controls and this response was completely inhibited by phloretin (500μM). Kinetic analysis of inside-out membranes enriched in ApUT showed ~28 fold increase in urea permeability (3.25 ± 0.37 x10-4 cm/s) compared to control vesicles (0.114 ± 0.023 x10-4 cm/s). Urea transport across the channel was phloretin inhibitable (75 % inhibition), and the site of inhibition may be located on the extracellular side. Glycerol and urea analogues such as methylamine, dimethylurea, formamide, acetamide, methylurea, propanamide and ethylamine did not permeate across ApUT. Furthermore, ApUT did not conduct protons or ammonia. Urea transport through ApUT shows saturable kinetics with a km of 357 mM. In addition to urea ApUT also conducts water. Furthermore, phloretin inhibition studies suggest that both urea and water permeates through the same pore.