Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat.