Airway epithelial ion transport is responsible for maintaining airway surface liquid composition and depth, an integral defence barrier in the lung. In Cystic Fibrosis, mutations in CFTR result in aberrant Cl- transport, resulting in airway dehydration, inflammation and infection. An alternative route for Cl- transport is the Calcium activated Cl- Channel (CaCC) (Ferrera et al., 2009). Our aim was to assess CaCC and CFTR-mediated Cl- secretion in the murine trachea using a perfusion potential difference model. Male Balb/c mice (20-25g,Charles River, UK) were anaesthetised (16mg/mL ketamine/ 2mg/mL xylazine i.p in water for injections), secured supine and tilted nose down. Solutions (all containing 1mM amiloride) were perfused at 25µL/min onto the tracheal lumen via a custom manifold, using a Pump 22 syringe pump (Harvard Apparatus, UK). Potential difference was measured using 3.5% agar/HBSS-bridge electrodes connected to calomel half cells in 2M KCl. The exploring electrode was placed in contact with the perfusate, the reference subcutaneously in the animals flank. The manifold had a 5cm extruded tubing output, placed 5mm into a transverse incision in the distal trachea. PD was measured via a high impedance voltmeter and Powerlab (World Precision Instruments, UK). Data are expressed as absolute or Δ mean TPD ± s.e.mean (mV). All Δ values are compared to PBS (initial study only), or Cl- free PBS baseline. For the initial study (n=9) solutions were serially perfused, determining a baseline PD (PBS Biosera, UK) of -1.5 mV ± 0.2 , then establishing a Cl- gradient (Cl- free PBS, Biosera, UK), which induced a depolarisation of Δ +1.4mV ± 0.2. Subsequent perfusion (in Cl- free PBS) of the CFTR blocker CFTRinh-172 (40µM, Merck, UK) further depolarised the PD (Δ +2.2 ± 0.2mV). Further perfusion of Cl- free PBS and CFTRinh-172 with 10mM Carbachol (Sigma-Aldrich, UK) an M3 muscarinic agonist, induced a hyperpolarisation of Δ -3.4 ± 0.5mV. Finally, 50µM CaCCinh-A01, a CaCC inhibitor (De La Fuente et al., 2008), partially inhibited the carbachol-induced hyperpolarisation (Δ +0.4 ± 0.5mV, n=9). All solutions contained 0.45% DMSO. In order to stimulate CFTR activity, (Salinas et al., 2004) 10µM Forskolin was perfused (in Cl- free PBS) inducing a hyperpolarisation Δ -2.1 ± 0.4mV (n=7, 0.6% DMSO), which was partially inhibited by 40µM CFTRinh-172 (Δ -1.5 ± 0.5mV). In a separate study, 10µM forskolin (in Cl- free PBS, 0.6% DMSO, n=7) induced a hyperpolarisation (Δ -1.7 ± 0.3mV) which was inhibited by 100µM CaCCinh-A01 (Δ -0.6 ± 0.3mV). The hyperpolarisation induced by carbachol, which will cause a rise in intracellular calcium, in the presence of CFTRinh-172, indicates that murine tracheal Cl- transport is largely CaCC mediated. Furthermore, our results indicate that the forskolin response is mainly CaCC rather than CFTR mediated in the murine trachea.