NHE3 is the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3(-/-) mouse with greatly reduced proximal tubule HCO(-)(3) absorption compared with NHE3(+/+) animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule HCO(-)(3) reabsorption in NHE3(-/-) mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of HCO(-)(3) (J(HCO3)) and fluid absorption (J(v)) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 microM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J(HCO3) and J(v) in NHE3(+/+) mice but failed to inhibit J(HCO3) or J(v) in NHE3(-/-) mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 microM bafilomycin caused a similar absolute decrement in J(HCO3) in wild-type and NHE3 null mice, indicating equivalent rates of HCO(-)(3) absorption mediated by H(+)-ATPase. Addition of 10 microM Sch-28080 did not reduce J(HCO3) in either wild-type or NHE3 null mice, indicating lack of detectable H(+)-K(+)-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating HCO(-)(3) reabsorption in the proximal tubule. A significant component of HCO(-)(3) reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H(+)-ATPase, but its activity is not significantly upregulated in NHE3 null mice.