To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, the membrane voltage (V_m), intracellular concentrations of Ca²⁺, Na⁺ ([Na⁺]_c), Cl^-, and H⁺ (pH_i) were measured in rat submandibular gland acini (RSMGAs). After a transient depolarization induced by a short application of acetylcholine (ACh, 5 µM, 20 s), RSMGAs showed strong delayed hyperpolarization (V_h,ACh, -95±1.8 mV) that was abolished by ouabain. In the HCO₃ ^--free condition, the V_h,ACh was also blocked by bumetanide, a blocker of Na⁺-K⁺- 2Cl^- cotransporter (NKCC). In the presence of HCO₃ ^- (24 mEq, bubbled with 5% CO₂), however, the V_h,ACh was not blocked by bumetanide but it was suppressed by ethylisoprolpylamiloride (EIPA), a Na⁺/H⁺ exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na⁺]_c was totally blocked by bumetanide in the absence of HCO₃ ^-, but only by half in the presence of HCO₃ ^-. ACh induced a prominent acidification of pH_i in the presence of HCO₃ ^-, and the acidification was further increased by EIPA treatment. Without HCO₃ ^-, an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH_i during the application of NH₄ ⁺ (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO₃ ^-. In summary, the ACh-induced anion secretion in RSMGAs is followed by the activation of NKCC and NHE, resulting an increase in [Na⁺]_c. The intracellular Na⁺-induced activation of electrogenic Na⁺/K⁺-ATPase causes V_h,ACh. The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO₃^-.