The present study investigated the inhibitory effect of extracellular ATP on Na absorption and the possible underlying mechanism in cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique. The cultured epithelia exhibited a Na -dependent basal current that could be predominately blocked by the epithelial Na channel (ENaC) blocker, amiloride (10 μM). Apical addition of ATP (10 μM) induced a reduction in basal I_SC. However, in the presence of amiloride or when apical Na was removed, the ATP-induced reduction was abolished and an increase in the I_SC was observed with kinetic characteristics similar to those reported previously for the ATP-induced Cl⁻ secretion, indicating that ATP could induce both Cl⁻ secretion and inhibition of Na absorption. Further reduction in I_SC after ATP challenge could be obtained with forskolin (10 μM), which indicates that different inhibitory mechanisms are involved. The ATP-induced inhibition of Na absorption, but not that induced by forskolin, could be abolished by the P₂ receptor antagonist, reactive blue (100 μM), indicating the involvement of a P₂ receptor in mediating the ATP response. ATP and uridine 5′-diphosphate (UDP; 100 μM), a relatively selective agonist for the pyrimidinoceptor, induced separate I_SC reduction, and distinct I_SC increases in the presence of amiloride, regardless of the order of drug administration, indicating the involvement of two receptor populations. The ATP-induced inhibition of Na absorption was mimicked by the Ca² ionophore, ionomycin (1 μM), whereas the Ca² chelators, EGTA and BAPTA-AM, abolished the ATP-induced, but not the forskolin-induced, inhibition of Na absorption, suggesting the involvement of a Ca² -dependent pathway. In the presence of the Cl⁻ channel blocker, DIDS (100 μM), both inhibitory and stimulatory responses to ATP were abolished, suggesting the involvement of a Ca² -activated Cl⁻ channels (CaCCs) in mediating both ATP responses. The ATP-induced as well as the forskolin-induced reduction in I_SC was not observed when Cl⁻ was removed from the bathing solution, indicating that Cl⁻ permeation is important for the inhibition of Na absorption. The results suggest the presence of a Ca² -dependent ENaC-inhibiting mechanism involving CaCC in mouse endometrial epithelial cells. Thus, extracellular nucleotides may play an important role in the fine-tuning of the uterine fluid microenvironment by regulating both Cl⁻ secretion and Na absorption across the endometrium.