The Calu-3 cell line is being investigated as a model for human submucosal gland serous cells. In a previous investigation of basal short-circuit current (I _sc) in Calu-3 cells, high levels of bumetanide-insensitive basalI _sc (∼60 μA/cm²) were measured in cells grown at an air interface. BasalI _sc was reduced only 7% by bumetanide, and the largest component of basalI _sc required both Cl⁻ and in the bathing solutions. BecauseI _sc could be partially inhibited by basolateral 4,4′-dinitrostilbene-2,2′-disulfonic acid and because the only known apical exit pathway for anions is the cystic fibrosis transmembrane conductance regulator, which has a relatively poor conductance for , it was concluded that most basalI _sc is-dependent Cl⁻ secretion [M. Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L690–L698, 1997]. We have now measured isotopic fluxes of³⁶Cl⁻and²²Na⁺across short-circuited Calu-3 cells and found that virtually none of the basal I _sc is Cl⁻ secretion or Na⁺ absorption. Thus, in contrast to the earlier report, we conclude that the major component of basalI _sc is secretion. Stimulation recruits primarily Cl⁻ secretion, as previously proposed.