Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO⁻₃ by a Cl ⁻-independent, serosal Na⁺-dependent, serosal bumetanide-insensitive, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca²⁺-activated K⁺ channels, reduced HCO⁻₃ secretion and caused the secretion of Cl ⁻ by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K⁺ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K⁺ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO⁻₃ secretion to Cl ⁻ secretion because the uptake of HCO⁻₃ across the basolateral membrane is mediated by a 4,4 ′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive Na⁺:HCO⁻₃ cotransporter. Since the stoichiometry reported for Na ⁺:HCO⁻₃ cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO⁻₃ entry and favor the secretion of Cl ⁻. Therefore, differential regulation of the basolateral membrane K⁺ conductance by secretory agonists could provide a means of stimulating HCO⁻₃ and Cl ⁻ secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO⁻₃ and a Cl ⁻ channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO⁻₃ secretion in the human airways warrants greater attention.