Although earlier work has pointed to the presence of Na /H exchangers (NHEs) in the rat epididymis, little is known about the regional distribution of various NHE isoforms and their functions. In the present work, expression of different isoforms of NHE in cultured epithelia of the efferent duct and cauda epdidymidis were studied. Reverse transcription-polymerase chain reaction revealed the presence of NHE1, NHE2, and NHE3, but not NHE4, message in both cultured epithelia. Western blot analysis detected the presence of NHE1 and NHE2 proteins in both cultured epithelia, but NHE3 protein was only detected in the cultured epithelial cells from the efferent duct. Immunohistochemical studies demonstrated that NHE2 was localized in the cytoplasm of the ciliated cells, whereas NHE3 was localized at the apical membrane of the principal cells of the efferent duct. The NHE activities in both cultured epithelia were inhibited by 10 μM HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphonate), a NHE1 inhibitor, by approximately 76%. The HOE-694-resistant NHE activities in the cultured epithelia of efferent duct and cauda epididymidis were completely inhibited by 20 μM S3226 (3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride), a NHE3 inhibitor, and 300 μM HOE-694 (a dose that can completely block NHE2), respectively. These results indicated that NHE1, NHE2, and NHE3 were expressed in the cultured epithelial cells of the efferent duct, whereas only NHE1 and NHE2 were expressed in the cultured epithelial cells of the cauda epididymidis. It is suggested that NHE1 may provide “housekeeping” functions in both epithelia, whereas NHE2 in the cauda epididymidis and NHE3 in the efferent duct may be involved in Na reabsorption and regulation of pH of the luminal fluid.