The Rhesus (Rh) family is composed of RhD and RhCE proteins, and of RhAG, RhCG and RhBG glycoproteins. Sequence analysis shows that this family belongs to the superfamily of ammonium transporters, which includes AMT (AMmonium Transporters) and MEP (MEthyl Permeases), which are families of proteins from plants and yeasts. A first experimental study that gave evidence that RhAG (the Rh glycoprotein specifically expressed in erythroid cells) and RhCG (an epithelial Rh glycoprotein, particularly expressed in the distal nephron) are also ammonium transporters was obtained in yeast: RhAG and RhCG transfection in MEP-deficient yeasts restore their growth in ammonium-containing medium (Marini et al. 2000). From this study was raised the question Rh glycoproteins acting as ammonium transporters in mammals, whereas it was it was accepted that transmembrane NH4+ transport is mainly mediated by K+-transporting systems, and that non-ionic diffusion is responsible for NH3 transport through biological membranes (despite a few challenging reports, Cooper et al. 2002). In this context, several laboratories began to investigate the functional role of Rh glycoproteins in vetebrate cells: are they ammonium transporters? If so, what is the transported species (NH3 or NH4+)? Up to now, functional results converge to conclude that Rh glycoproteins are indeed ammonium transporters. But, as it is the case for the various AMT and MEP proteins, the transported species (and the mode of transport) is still debated: NH3 transport, NH4+ transport, NH3 and NH4+ transport, or NH4+ and H+ exchange. In our laboratory, we have studied the functional role of human RhAG and RhCG after their heterologous expression in two different expression systems: HeLa cells and Xenopus laevis oocytes. Changes in intracellular pH (pHi) during exposure to an ammonium-containing solution (NH4Cl in the millimolar range) were monitored using video-imaging system coupled to the fluorescence of the pH-sensitive probe BCECF or using pH-selective microelectrodes. Analysis of ammonium-induced pHi changes indicates that these cells differed in their endogenous membrane NH3 and NH4+permeabilities: whereas HeLa cells exhibit a sudden huge alkalinization (related to NH3 influx into the cell, and its protonation) followed by a teeny tendency of pHi to recover (secondary acidification related to NH4+ influx into the cell, and its partial dissociation), X. laevis oocytes exhibit a barely visible increase in pHi, rapidly blunted by a large cell acidification. These results are consistent with a large NH3 but a low NH4+ membrane permeability in HeLa cells, but the opposite in X. laevis oocyte. By measuring ammonium-induced pHi changes in transfected cells, we observed that expressing RhAG and RhCG enhance both initial alkalinization and secondary acidification induced by ammonium exposure, consistent with the enhancement of ammonium influx (NH3 + NH4+) in a non-synchronic way (NH3 then NH4+ influx). Interestingly, sub-millimolar (100 to 1000 microM) ammonium concentrations induced inward currents in voltage-clamped (Vc = – 50 mV) RhAG- and in RhCG- expressing oocytes. Further analysis of the ammonium-induced current is consistent with NH4+-related current, depending on Rh-induced NH3 transport. Taken together, these results show not only that RhAG and RhCG are ammonium transporters, but also that these human proteins are promoting the transmembrane transport of NH3 and of NH4+ (Bakouh et al. 2004; Benjelloun et al. 2005).