ABSTRACT Urea transporters (UTs) are a family of urea-selective channel proteins that play an essential role in the urine-concentrating mechanism of the mammalian kidney. In addition to urea, UT-A2 and UT-A3 – the N- and C-terminal regions of full-length UT-A1, respectively – and UT-B transport water, and human UT-B transports water and ammonia (NH3). However, UT-A-mediated NH3 transport has not been evaluated. Given that regulated renal NH3/NH4+ transport by renal epithelial cells is essential to acid–base homeostasis and considering UT-A2 and UT-A3 localization in the inner medulla, where the transport of urea, water, and NH3 is important, it is plausible that UT-A-mediated NH3 transport could be physiologically relevant. The present study characterized the urea, water, and NH3 transport properties and solute pathways of murine UT-A2, UT-A3, and UT-B heterologously expressed in Lithobates catesbeianus oocytes. Control and UT-expressing oocytes were evaluated for surface protein expression through lysine-biotinylation and immunoblotting. Urea uptake was measured using radiolabeled urea, water permeability was assessed using video microscopy, and NH3 transport was monitored using a surface pH microelectrode. All UT-encoding cRNAs were translated, glycosylated, and inserted into the oocyte membrane. Wild-type UT-expressing oocytes displayed significantly higher urea, water, and NH3 transport than day-matched water-injected control cells. Pre-treating the oocytes with phloretin or mutating the urea pore threonines (Thr177 and Thr339 human UT-B numbering) to valines (Val) attenuated UT-mediated urea, water and NH3 transport to control oocyte values. Our study showed for the first time that UT-A2 and UT-A3 increase the membrane NH3 permeability. Thus, besides the critical role of UTs in urinary concentration, these proteins may also impact acid–base homeostasis and contribute to other processes associated with health and disease.