Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)–dependent Cl⁻ conductance. Activation of Cl⁻ secretion in airways depends on simultaneous activation of luminal Cl⁻ channels and basolateral K⁺ channels. We determined the role of basolateral K⁺ conductance in cAMP- dependent Cl⁻ secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na⁺ conductance and defective cAMP-mediated Cl⁻ conductance. In non-CF tissues, Cl⁻ secretion was significantly inhibited by the chromanol 293B (10 μ mol/liter), a specific inhibitor of K_VLQT1 K⁺ channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba^{2 +} (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 μ mol/liter) simultaneously activated Cl⁻ and K⁺ conductance. The K⁺ conductance was reversibly inhibited by Ba^{2 +} and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K_VLQT1. We conclude that the K⁺ channel K_VLQT1 is important in maintaining cAMP-dependent Cl⁻ secretion in human airways. Activation of K_VLQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl⁻ channel activity or alternative Cl⁻ channels could help to circumvent the secretory defect.