Rhcg and the H-ATPase B1 subunit are co-expressed in renal acid-secretory intercalated cells. We examined the role of Rhcg in ammonium (NH₄) handling and its involvement in proton secretion by the collecting duct (CD). Genetic mutations or ablation of the B1 subunit cause distal renal tubular acidosis (dRTA). Rhcg+/+, +/­, and -/- and B1-/- mice and their controls (C) were studied in metabolic cages, under baseline (BL) and after an acute (AAL) or a chronic (CAL) HCl-load. Under BL, no difference in blood and urine parameters was observed, only B1-/- mice exhibited alkaline urine pH. During AAL, Rhcg-/- mice showed severe metabolic acidosis (MA) with a decrease in blood pH and HCO₃ concentration. They excreted 50% less NH₄ than C concomitant with a more alkaline urine pH. Most of Rhcg-/- mice died before the end of CAL due to low NH₄ excretion and massive dehydration. During CAL, Rhcg+/­ and B1-/- mice exhibited also MA with lower blood pH and HCO₃ concentration and a more alkaline urine as compared to C. Only B1-/- mice excreted less NH₄ than C while Rhcg+/­ mice decreased their titratable acid excretion. Finally, we assessed luminal ammonia membrane permeability (MP) and proton (H) secretion in microperfused CD coupled to B1 and Rhcg mRNA quantification in whole kidneys. After an AAL, NH₃ MP and H secretion were reduced in CD from Rhcg-/- mice. Renal Rhcg mRNA was totally not detectable while there was no difference in renal B1 mRNA expression. Also Rhcg+/­ mice had lower Rhcg mRNA expression, and reduced H and MP values. Renal Rhcg mRNA was decreased in B1-/- mice. Moreover, in CD from C mice, pharmacological H-ATPase inhibition not only blocked H-secretion but also reduced NH₃ MP. Thus, Rhcg is necessary to excrete acid. The metabolic similarities between Rhcg+/­ and B1-/- mice and the H secretion defect observed in Rhcg-/- mice suggest a direct functional link between Rhcg and H-ATPases for final acid excretion.