In cystic fibrosis respiratory epithelial cells, the absence or dysfunction of the chloride channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator) results in reduced chloride ion transport. In contrast, Ca2+-stimulated Cl- secretion is intact in cystic fibrosis airway epithelia. One possible target for drug discovery aiming at treating cystic fibrosis is to correct the ionic imbalance through stimulation of alternative ionic pathways that may compensate the failure of epithelial Cl- conductance. Here, using a simple high-throughput screening assay to search for Cl- channels modulators in the cystic fibrosis nasal epithelial cell line JME-CF15, the compound guanabenz (Wytensin), an alpha2-selective adrenergic agonist was found positive. Using iodide effluxes and electrophysiological recordings, we showed that guanabenz-activated (EC50=831 nM) a DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) sensitive and Ca2+ dependent Cl- channel (CaCC). Guanabenz activated a linear Cl- channel with unitary single-channel conductance of 8 pS. Recording calcium signals in CF15 cells showed that guanabenz increased the intracellular Ca2+ concentration stimulating an influx of Ca2+. In the absence of extracellular Ca2+, the guanabenz effects on Ca2+ influx and activation of CaCC were both abolished. These data demonstrate that guanabenz activates Ca2+-dependent Cl- channels via a Ca2+ influx in human cystic fibrosis airway epithelial cells.