Human airway epithelia express Ca2+-activated Cl−channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+channels in CaCC-mediated Cl−secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+channels. The K+channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl−secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl−secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl−secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.