The aims of the present study were to estimate the fraction of renal brush border membrane Na+-H+ exchange activity mediated by the isoform NHE3 and to evaluate whether the increased brush border Na+-H+ exchange observed in metabolic acidosis is due to increased expression of NHE3 protein. Compared with other isoforms, NHE3 is known to have a unique profile of sensitivity to pharmacologic inhibitors, including relative resistance to amiloride analogs and HOE694. We therefore assessed the inhibitor sensitivity of pH gradient-stimulated 22Na uptake in renal brush border vesicles isolated from normal rats. The I50 values for amiloride (30 microM), dimethylamiloride (10 microM), ethylisopropylamiloride (6 microM), and HOE694 (>100 microM) were markedly dissimilar from those reported for NHE1 and NHE2 but were nearly identical to reported values for NHE3. Na+-H+ exchange activity in renal brush border vesicles isolated from rats with 5 days of NH4Cl-induced metabolic acidosis was increased 1.5-fold compared with control rats, with no change in inhibitor sensitivity. Western blot analysis indicated that NHE3 protein expression was greater in brush border membranes from acidotic compared with control rats. We conclude that virtually all measured Na+-H+ exchange activity in brush border membranes from control and acidotic rats is mediated by NHE3 and that metabolic acidosis causes increased expression of renal brush border NHE3 protein.