Mice with a targeted disruption of Na+/H+exchanger NHE-3 gene show significant reduction in[Formula: see text] reabsorption in proximal tubule, consistent with the absence of NHE-3. Serum[Formula: see text], however, is only mildly decreased (P. Schulties, 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), indicating possible adaptive upregulation of[Formula: see text]-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 −/−) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2(net[Formula: see text] flux, JtCO2) was measured in the presence of 10 μM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 μM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD, JtCO2increased in NHE-3 mutant mice (3.42 ± 0.28 in WT to 5.71 ± 0.39 pmol ⋅ min−1⋅ mm tubule−1in mutants, P < 0.001). The SCH-sensitive net[Formula: see text] flux remained unchanged, whereas the DES-sensitive [Formula: see text] flux increased in the CCD of NHE-3 mutant animals. In OMCD, JtCO2increased in NHE-3 mutant mice (8.8 ± 0.7 in WT to 14.2 ± 0.6 pmol ⋅ min−1⋅ mm tubule−1in mutants, P < 0.001). Both the SCH-sensitive and the DES-sensitive [Formula: see text] fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl−/[Formula: see text]exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate that[Formula: see text] reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatory[Formula: see text] reabsorption in NHE-3-deficient animals.