Airway submucosal glands are important sites of cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl(-)) channel expression and fluid secretion in the airway. Whereas both mouse and human submucosal glands and their serous acinar cells express CFTR, human glands and serous cells secrete much more robustly than mouse cells/glands in response to cAMP-generating agonists such as forskolin and vasoactive intestinal peptide. In this study, we examined mouse and human serous acinar cells to explain this difference and reveal further insights into the mechanisms of serous cell secretion. We found that mouse serous cells possess a robust cAMP-activated CFTR-dependent Cl(-) permeability, but they lack cAMP-activated calcium (Ca(2+)) signaling observed in human cells. Similar to human cells, basal K(+) conductance is extremely small in mouse acinar cells. Lack of cAMP-activated Ca(2+) signaling in mouse cells results in the absence of K(+) conductances required for secretion. However, cAMP activates CFTR-dependent fluid secretion during low-level cholinergic stimulation that fails to activate secretion on its own. Robust CFTR-dependent fluid secretion was also observed when cAMP stimulation was combined with direct pharmacological activation of epithelial K(+) channels with 1-ethyl-2-benzimidazolinone (EBIO). Our data suggest that mouse serous cells lack cAMP-mediated Ca(2+) signaling to activate basolateral membrane K(+) conductance, resulting in weak cAMP-driven serous cell fluid secretion, providing the likely explanation for reduced cAMP-driven secretion observed in mouse compared with human glands.