The erythrocyte Rh and Rh-associated (RhAG) proteins have distant sequence identity to a family of ammonium transporters found in yeast and bacteria. We previously showed that RhAG mediates movement of ammonium when expressed in yeast and in Xenopus oocytes. Importantly, these are the first mammalian proteins found to transport ammonium as a principal substrate. Elucidation of the mechanism and actual substrate(s) transported (protonated NH4+ or unprotonated NH3, or both) is important to understand their role in elimination of ammonium, proton recycling, and their impact on cellular pH and acid/base regulation. Functional characterization revealed that uptake was independent of the membrane potential and the Na+ gradient, but was dramatically stimulated by raising extracellular pH or lowering intracellular pH. This suggested that uptake was coupled to an outwardly directed H+ gradient and led us to hypothesize that RhAG might function by an H+-coupled, counter-transport mechanism. To further define the mechanism and actual substrate transported, RhAG-expressing oocytes were exposed to varying concentrations of NH4+ with constant NH3, and vice versa, by manipulation of the NH4Cl concentration and the pH of the buffer. A voltage-ramping protocol was used to evaluate changes in membrane conductance and reverse potential to measure membrane depolarization. Radioactive flux uptake of 14C-methylammonium, an analogue of ammonium, was used to measure transport. In the presence of substrate in the physiologic range (20 uM-500 uM), RhAG-mediated transport responded to the concentration of protonated NH4+ rather than the amount of unprotonated NH3 present. Currents in RhAG-expressing oocytes did not differ from water-injected controls. No significant changes in membrane conductance or membrane depolarization and reverse potential were observed. Taken together these data support a role for RhAG in the electronuetral transport of NH4+ by exchange with H+, and for erythrocytes in the maintenance of total blood ammonia levels. Sequestration of ammonium by erythrocytes would keep blood plasma levels low, preventing exposure of cells to toxic levels. Erythrocytes are ideally postioned to then transport ammonium to be exchanged in the liver and kidney, where other Rh-related proteins (RhBG and RhCG) are expressed.