Salivary gland acinar cells express both intermediate- and large-conductance Ca²⁺-activated K⁺ channels, often termed IK (Sk4) and BK (Slo) channels, which are encoded by the Kcnn4 and the Kcnma1 genes, respectively. To test the roles of these channels in submandibular glands, we examined the functional consequences of null mutations in the Kcnn4 and the Kcnma1 genes. As anticipated, we found that functional IK1 and BK channels were absent in submandibular acinar cells from Kcnn4^-/- and Kcnma1^-/- mice, verifying their genetic identities. The cytoplasmic Cl^– concentration of salivary acinar cell is approximately 4- to 5-fold higher than the Cl^– electrochemical equilibrium. Thus, when Ca²⁺-mobilizing receptor agonists stimulate the apical Ca²⁺-activated Cl^– channel, Cl^– exit occurs into the lumen and Na⁺ and water follow. However, Cl^– exit also depolarizes the acinar cell, and if the membrane potential approached the Cl^– equilibrium potential, net Cl^– movement and fluid secretion would stop. Secretion depends, therefore, on the maintenance of a membrane potential (V_m) negative to the Cl^– reversal potential. This membrane hyperpolarization has been linked to IK1 and/or BK channels. As expected, stimulation of acinar cells from wildtype mice with the muscarinic receptor agonist carbachol (0.3 µM) produced a rapid depolarization to near the Cl^– reversal potential, but the V_m quickly returned to a point approximately midway (-55±2 mV) between the equilibrium potentials for K⁺ (E_eq=-85 mV) and Cl^– (E_eq=-24 mV). Unexpectedly, submandibular acinar cells from either Kcnn4^-/- or Kcnma1^-/- mice maintained a relatively hyperpolarized V_m during muscarinic receptor stimulation (-51±3 and -48±3 mV for Kcnn4^-/- and Kcnma1^-/-, respectively) – values comparable to that seen in wildtype mice. Consistent with this observation, fluid secretion induced in vitro by 0.5 µM carbachol was not inhibited in mice lacking expression of either IK1 or BK channels in isolated, perfused submandibular glands. These results indicate that in isolation, either IK1 or BK channels can maintain the hyperpolarized V_m required for normal sustained fluid secretion. In contrast, the V_m of acinar cells in double Kcnn4^-/-/Kcnma1^-/- mice was significantly depolarized (-35±2 mV), and fluid secretion was markedly reduced (~70%). Thus, in the absence of both IK1 and BK channels, acinar cell V_m remained sufficiently hyperpolarized to support fluid secretion, but at a significantly reduced rate. We found that inhibition of the electrogenic Na⁺,K⁺-ATPase produced little, if any, depolarization during muscarinic receptor stimulation in wildtype acinar cells (-51±5 mV), whereas simultaneous inhibition of both Ca²⁺-activated K channels and the ATPase depolarized cells to near the Cl^– reversal potential (-27±4 mV). Taken together, our results demonstrate that IK1 and BK channels play major roles in submandibular acinar cell function, and under severe pathological conditions (such as loss of expression of IK1 and BK channels) Na⁺,K⁺-ATPase has a limited capacity to hyperpolarize V_m sufficiently to support secretion.