The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant and microbial membranes. A bacterial homolog, AmtB, forms a channel-like structure and appears to function as a NH3 gas channel. To evaluate the function of eukaryotic homologs, human RhCG glycoproteins and plant ammonium transporters were expressed and compared in Xenopus oocytes and yeast. Rh glycoproteins mediated saturable electroneutral transport of methylammonium (MeA). Transport was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in Rh glycoprotein-expressing oocytes. In contrast, expression of plant AMTs induced pHo-independent MeA+ uptake and specific NH4+- and MeA+-currents. These currents were distinct from endogenous currents. The different mechanisms of transport were also observed after heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner: while plant AMTs specifically transport NH4+, or co-transport NH3/H+, Rh glycoproteins mediate electroneutral NH3 transport.