Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10−7 M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl− secretion ( ICl) beginning after 3–10 min, reaching a plateau after 20–45 min at Δ ICl = 15–50 μA/cm2. Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated ICl in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1β (produced by both epithelia and neutrophils during infections) stimulated ICl similar to flagellin. Flagellin-, IL-1β-, ATP-, and forskolin-stimulated ICl were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1β altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-κB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1β-stimulated ICl by 33–50% but had smaller effects on IL-8 and NF-κB. It is concluded that: 1) flagellin and IL-1β activated p38, NF-κB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating ICl and IL-8 but not NF-κB; and 3) p38 was more important in flagellin- than IL-1β-stimulated responses. During P. aeruginosa infections, flagellin and IL-1β are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-κB, and IL-8 would persist.