Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (DeltaF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH(2)-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial "internalization platform" involving both caveolin-1 and functional, laterally mobile CFTR.