The present study investigated the involvement of Na -HCO₃⁻ cotransporter in mediating cAMP-stimulated HCO₃⁻ secretion across the cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique and intracellular pH measurement. Forskolin stimulated a rise in the I_SC, 55.6% and 52.1% of which could be reduced by the removal of extracellular Cl⁻ or by eliminating the contribution of Cl⁻ secretion by bumetanide, an inhibitor of Na -K -2Cl⁻ cotransporter, respectively. More than 80% reduction in the forskolin-induced I_SC was obtained when both Cl⁻ and HCO₃⁻ in the bath were removed or in HCO₃⁻-free solution with bumetanide, indicating that the I_SC depended on both Cl⁻ and HCO₃⁻. The presence of the Na channel-blocker amiloride in the apical solution did not reduce the forskolin-induced I_SC; however, the I_SC could be abolished by removing Na from the bathing solution, suggesting that the Cl⁻- and HCO₃⁻-dependent I_SC was also dependent on basolateral Na . The forskolin-stimulated I_SC could be reduced 43.6% by removal of HCO₃⁻ and 47.9% by a Na -HCO₃⁻-cotransporter inhibitor, dihydrogen-4,4′-didsothiocyanostilbene-2,2′-disulfonic acid (H₂DIDS). The inhibitory effect of H₂DIDS was observed in Cl⁻-free solution, but not when HCO₃⁻ was removed, thus confirming its effect on HCO₃⁻-dependent transport. Intracellular pH measurements demonstrated that the recovery from cellular acidification depended on the presence of both basolateral Na and HCO₃⁻, further indicating the involvement of Na -HCO₃⁻ cotransporter. Reverse transcription-polymerase chain reaction experiments confirmed the expression of Na -HCO₃⁻ cotransporter in the mouse endometrium. The results suggest that basolaterally located Na -HCO₃⁻ cotransporter is involved in mediating cAMP-stimulated HCO₃⁻ secretion across the mouse endometrial epithelium.