The luminal fluid microenvironment of the uterus is important for sperm capacitation and embryo development. In an attempt to understand the possible role of Na /H exchangers (NHEs) in uterine function, the mRNAs of different NHE isoforms as well as their subcellular localization (apical versus basolateral) and functional activity were investigated in mouse endometrial epithelial cells using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and intracellular pH (pH_i) measurement techniques. The presence of NHE1, NHE2, and NHE4, but not NHE3 mRNAs were revealed by RT-PCR. Immunostaining showed that NHE1, NHE2, and NHE4 were present in both apical and basolateral membranes. The pH_i recovery from intracellular acidification was Na -dependent; however, the rate of pH_i recovery depending on basolateral Na was 12.4 times faster than that depending on apical Na . The Na -dependent rate of pH_i recovery was also inhibited by amiloride, indicating H extrusion through NHEs; however, the amiloride sensitivity of the apical membrane was less than that of the basolateral membrane, suggesting the involvement of different types of NHEs in the two membranes. The results indicate that the basolaterally located NHE1, NHE2, and NHE4, in addition to participating in the homeostatic control of intracellular pH, may play a role in H extrusion in order to achieve transepithelial HCO₃⁻ secretion. The apically located NHEs may be involved in mediating Na absorption as alternatives of or complementary to epithelial Na channels.