Objectives/Hypothesis: The cystic fibrosis transmembrane conductance regulator (CFTR) serves as a predominant Cl- transport conduit in airway epithelium and is inhibited by cigarette smoke in vitro and in vivo. Activation of secondary Cl- transport pathways through calcium-activated Cl- channels (CaCC) has been postulated as a mechanism to bypass defects in CFTR-mediated transport. Because it is not known whether CaCCs are also inhibited by tobacco exposure, the current study was designed to investigate the effect of cigarette smoke condensate (CSC) on CaCC transport. Study Design: In vitro study. Methods: Well-characterized primary murine nasal septal epithelial (MNSE) and human sinonasal epithelial (HSNE) cultures were exposed to CSC in Ussing chambers. We monitored CaCC short-circuit current through stimulation of P2Y purinergic receptors with uridine triphosphate or adenosine triphosphate and selective inhibition of the CFTR-dependent secretory pathway. Characterization of CaCC current was also accomplished in primary airway cells derived from transgenic CFTR-/- (knockout) murine models. Results: Change in CaCC-mediated current (ΔISC representing transepithelial Ca-mediated Cl- secretion in μA/cm2) was significantly decreased in CSC-exposed wild type MNSE when compared to controls (32.8 ± 4.6 vs. 47.5 ± 2.3; respectively; P < .02). A similar effect was demonstrated in CFTR -/- MNSE cultures (33.4 ± 2.8 vs. 38.6 ± 2.0; P < .05>. HSNE cultures also had a significant reduction in ISC (16.1 ± 0.6 vs. 22.7 ± 0; P = .008). Conclusions: CSC affects multiple pathways fundamental to airway ion transport, including both cyclic adenosine monophosphate and calcium activated Cl- transport. Inhibition of Cl- transport may contribute to common diseases of the airways, such as chronic rhinosinusitis and chronic obstructive pulmonary disease. © 2010 The American Laryngological, Rhinological and Otological Society, Inc.