The mucosa of the proximal airways defends itself and the lower airways from inhaled irritants such as capsaicinoids, allergens, and infections by several mechanisms. Sensory nerves monitor the luminal microenvironment and release the tachykinin substance P (SP) to stimulate mucus secretion. Here, we have studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in SP stimulation by comparing mouse airway submucosal gland responses in wild-type (WT) and CFTR−/− mice. Capsaicinoids (chili pepper oil) increased fluid secretion by glands from WT mice five-fold, and this response was abolished by exposing the basolateral aspect of the tracheas to L-732,138 (10 μmol/l), a specific antagonist of the neurokinin-1 receptor. Secretion was also stimulated 25-fold by basolateral application of SP, and this response was strongly inhibited by the CFTR inhibitor CFTR_inh172. In contrast, submucosal glands from CFTR knockout mice failed to secrete when stimulated by SP (1 μmol/l), although those from wild-type control littermates were responsive. SP stimulation of wild-type glands was also abolished by clotrimazole (25 μmol/l), a blocker of Ca²⁺-activated K⁺ channels. These results indicate that SP mediates local responses to capsaicinoids through a mechanism involving coordinated activation of CFTR and K⁺ channels. To our knowledge, this is the first study in which CFTR-dependent responses to substance P have been directly demonstrated. Since CFTR regulation is qualitatively similar in human and mouse glands, loss of this local regulation in CF may contribute to reduced innate defenses in CF airways.