The Lake Magadi tilapia (Alcolapia grahami) is an unusual fish, excreting all its nitrogenous waste as urea because of its highly alkaline and buffered aquatic habitat. Here, using both physiological and molecular studies, we describe the mechanism of branchial urea excretion in this species. In vivo, repeated short-interval sampling revealed that urea excretion is continuous. The computed urea permeability of A. grahami gill is 4.74×10−5±0.38×10−5 cm s−1 (mean ± S.E.M., N=11), some 10 times higher than passive permeability through a lipid bilayer and some five times higher than that of even the most urea-permeable teleosts studied to date (e.g. the gulf toadfish). Transport of urea was bidirectional, as demonstrated by experiments in which external [urea] was elevated. Furthermore, urea transport was inhibited by classic inhibitors of mammalian and piscine urea transporters in the order thiourea>N-methylurea>acetamide. A 1700 base pair cDNA for a putative Magadi tilapia urea transporter (mtUT) was cloned, sequenced and found to display high homology with urea transporters from mammals, amphibians and other fishes. When cRNA transcribed from mtUT cDNA was injected into Xenopus laevis oocytes, phloretin-inhibitable urea uptake was enhanced 3.4-fold relative to water-injected controls. Northern analysis of gill, red blood cells, liver, muscle and brain using a portion of mtUT as a probe revealed that gill is the only tissue in which mtUT RNA is expressed. Magadi tilapia gill pavement cells exhibited a trafficking of dense-cored vesicles between the well-developed Golgi cisternae and the apical membrane. The absence of this trafficking and the poor development of the Golgi system in a non-ureotelic relative (Oreochromis niloticus) suggest that vesicle trafficking could be related to urea excretion in Alcolapia grahami. Taken together, the above findings suggest that the gills of this alkaline-lake-adapted species excrete urea constitutively via the specific facilitated urea transporter mtUT.