Na+/H+ antiporter activity in renal brush border vesicles from control rabbits and rabbits made acidotic with 3 days of NH4Cl gavage was measured using the acridine orange method. Acidotic rabbits exhibited a significantly higher V(max) for antiporter activity (2.80 ± 0.45 fluorescence units.s-1.mg protein-1) compared with controls (1.31 ± 0.13) but the K(m) for Na+ was unchanged (23.7 ± 3.5 for acidotic, 19.1 ± 3.2 mM for controls). When the V(max) for Na+/H+ antiporter activity was considered in relation to the degree of acidosis achieved in the experimental animals, there was a correlation (r = -0.75) between V(max) and plasma total CO2 concentration. Amiloride (100 μM) inhibited Na+/H+ exchange (Na+ = 90 mM) by 59 ± 7% in both control and acidotic animals, indicating that the observed stimulation in Na+/H+ antiporter activity was not due to increased electrically coupled cation exchange. These findings suggest that the response of the proximal tubule to chronic metabolic acidosis involves an adaptive increase in the V(max) for Na+/H+ antiporter activity in the brush border membrane that is correlated to the degree of acidosis in the animals.