The Na⁺/H⁺ exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H⁺-activated ²²Na uptake by 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 μM. Kinetic analyses showed that the extracellular Na⁺ dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H⁺ activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H⁺ with a half-maximal activation value of pK6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H⁺, suggesting that they may play distinct physiological roles in the kidney.