Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH3/NH4+ transport. The structure of Rhbg is not yet resolved however a high-resolution crystal structure of AmtB, a bacterial homologue of Rh, has been determined. We aligned the sequence of Rhbg to AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, Histidine-183 (H183), Histidine-342 (H342) and Tryptophan-230 (W230), within the hydrophobic pore where they presumably serve to control NH3 transport. A fourth residue, Phenylalanine-128 (F128) was positioned at the upper vestibule presumably contributing to recruitment of NH4+. We generated three mutations each of H183, H342, W230 and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH3/NH4+ and methyl amine/ammonium (MA/MA+)-induced currents, intracellular pH and surface pH (pHs) among oocytes expressing the mutants, Rhbg or injected with H2O. In H183 and W230 mutants, NH4+-induced current and intracellular acidification were inhibited compared to Rhbg and MA-induced intracellular alkalinization was completely absent. Expressing H183A or W230A mutants inhibited NH3/NH4+- and MA/MA+-induced decrease in pHs to the level observed in H2O-injected oocytes. Mutations of F128 did not affect transport of NH3 or NH4+ significantly. These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter.