The ammonium transport function of ghosts from human (Rh_null , CO_null ) and mouse (KO Rh and Rhag) variants were analyzed on the stopped-flow spectrofluorometer. Previous studies with normal ghosts exposed to ammonium gradients have shown a rapid alkalinization attributed to NH₃ influx, followed by a slower acidification phase that involved the anion exchanger Band 3, which might have potentially reduced alkalinization rate. To minimize these factors alkalinization rate constants analysis was performed in the absence of chloride (replaced by potassium gluconate) and to better estimate ammonium fractions crossing the red cell membrane via the protein pathway and lipid diffusion, we used gluconate salts of ammonium, methylammonium and of hydrazine to generate 20 mM gradients. In these conditions, kinetics of alkalinization were rapid and essentially monophasic. Analysis of rate constants indicate that RhAG proteins are major ammonium transporters of red cells and that NH₃ conductance of AQP1 might be only 10% that of RhAG.