The proximal convoluted tubule (PCT) reabsorbs most of the filtered bicarbonate. Proton secretion is believed to be mediated predominantly by an apical membrane Na⁺/H⁺ exchanger (NHE). Several NHE isoforms have been cloned, but only NHE3 and NHE2 are known to be present on the apical membrane of the PCT. Here we examined apical membrane PCT sodium-dependent proton secretion of wild-type (NHE3^+/+/NHE2^+/+), NHE3^–/–, NHE2^–/–, and double-knockout NHE3^–/–/NHE2^–/– mice to determine their relative contribution to luminal proton secretion. NHE2^–/– and wild-type mice had comparable rates of sodium-dependent proton secretion. Sodium-dependent proton secretion in NHE3^–/– mice was approximately 50% that of wild-type mice. The residual sodium-dependent proton secretion was inhibited by 100 μM 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Luminal sodium-dependent proton secretion was the same in NHE3^–/–/NHE2^–/– as in NHE3^–/– mice. These data point to a previously unrecognized Na⁺-dependent EIPA-sensitive proton secretory mechanism in the proximal tubule that may play an important role in acid-base homeostasis.