In recent work we identified the presence of an apical Na:H exchanger in macula densa cells. Since this antiporter is also commonly expressed at the basolateral membrane of renal epithelia, studies were performed to determine if this arrangement also occurs in macula densa cells. Using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pHi) of macula densa cells was measured with fluorescence microscopy using BCECF. N-methyl-D-glucamine and/or cyclamate were used to isosmotically substitute for Na and/or Cl in perfusion and bath Ringer's solutions. Control pHi, during perfusion with 25 mM NaCl and 150 mM NaCl in the bath, averaged 7.44 ± 0.04 (n = 30). Removal of Na from the bath (i.e. basolateral solution) decreased pHi by 0.65 ± 0.04 (n = 8; p < 0.01). Ethylisopropyl-amiloride (EIPA, 300 μM), an inhibitor of Na:H exchange, blocked Na-dependent acidification, whereas 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, 250 μM), an inhibitor of HCO3 transport systems, had no effect. Pharmacological analysis revealed a low affinity of this basolateral Na:H exchanger for EIPA (Ki = 9.0 μM), whereas the apical NHE was moderately sensitive to this drug (Ki = 0.86 μM). Phorbol 12-myristate 13-acetate (PMA, 100 nM), a known activator of protein kinase C, significantly reduced activity of the basolateral exchanger. The results of these studies suggest a unique configuration of NHEs in macula densa cells expressing NHE-3 isoform at the basolateral, and NHE-2 at the apical membrane.