Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+, K+, 2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1mM to 2M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of chloride ions from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor, furosemide (10-100µM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P<0.05). Localized tracheal pretreatment with the chloride channel inhibitors, DIDS or niflumic acid (100µM) also significantly reduced cough, whereas the GABA_A receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate chloride ions due to the expression of the furosemide-sensitive chloride transporter, NKCC1. Efflux of intracellular chloride, in part through calcium-activated chloride channels, may play an important role in regulating airway afferent neuron activity.