The present work examined the effects of chronic metabolic acidosis on the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) activity both in rat renal cortical homogenates and in their luminal membranes. Moreover, to assess the effect of acidosis on H+ transport by the apical H(+)-ATPase, we have developed a detergent-dilution procedure, resulting in the formation of sealed vesicles having this enzyme at their external surface. NH4Cl loading for 4 days had no effect on homogenates H(+)-ATPase activity, estimated with either N-ethylmaleimide or bafilomycin A1. In contrast, H(+)-ATPase activities were increased significantly by about 30% in both native apical membranes prepared by Ca2+ aggregation and detergent-treated luminal vesicles from acidotic animal. Kinetic analysis revealed that this stimulation was solely through changes in the Vmax for ATP. In membranes prepared by Mg2+ aggregation, acidosis also caused significant stimulation of the H(+)-ATPase activity. In addition, the initial rate of ATP-induced intravesicular acidification was 25% higher in reoriented H(+)-ATPase vesicles from acidotic rats, whereas passive proton permeability was identical in both groups. Finally, both vesicle enrichments and yields of luminal markers and de-enrichments and yields of intracellular membrane markers were identical in the two groups. These results provide enzymatic and functional evidence suggesting that chronic acidosis induces an adaptative change in the rat brush border H(+)-ATPase.