The Overhydrated Stomatocytic (OHSt) erythrocytes of four patients, characterized by the F65S mutation (Bruce et al, Blood 2009) located in the pore of the ammonia channel Rh Associated Glycoprotein (RhAG), were studied. We have previously demonstrated that the equivalent substitution (F74L) in the non-erythroid analogue RhCG resulted in a reduction of the ammonia influx of 50% in transfected HEK293 cells.Ghosts prepared by hypotonic lysis of OHSt and control (Ctl) erythrocytes, resealed in the presence of a pH-sensitive probe (pyranine), exhibited echinocytic morphology. Images of 1667 (Ctl) and 1998 (OHSt) echinocytes, visualized by light microscopy, allowed the determination of average ghost diameters: 5.81±0.2 µm (Ctl) and 5.83±0.06 µm (OHSt). RhAG densities, as determined by flow cytometry, were similar for Ctl and OHSt (78 000 to 80 000 copies/cell).Ammonium permeability (P’NH3) was measured by following the internal compartment alkalinisation, after submitting ghosts to inward ammonium gradients. Alkalinisation rate constants (k) were deduced from stopped-flow analysis of pH variations at 15°C, allowing the determination of P’NH3 by using the simplified equation P’NH3=k.r/3 (r: radius of spheric ghosts). The P’NH3 values were significantly different between Ctl and OHSt samples (1.7±0.11 vs 0.71±0.004 µm.s−1).Additionally, water permeability was measured in the same OHSt resealed ghosts but containing 8mM 6-carboxyfluorescein (6-CF), after submitting them to an osmotic gradient (150mosm/kg/H2O mannitol). The osmotic water permeability (Pf) values deduced from the fluorescence quenching at 15°C were similar between Ctl and OHSt samples (0.035±0.005 vs 0.035±0.04 cm.s−1).In conclusion, the F65S mutation of RhAG induces a reduction of ammonia flux resulting in an alteration of pHi regulation. The decrease of ammonia conductance through the mutated RhAG can result from loss of hydrophobicity and/or from structural modifications inside the pore of the channel.