P. aeruginosa is primarily an extracellular pathogen which adheres to cystic fibrosis epithelial cells due to increased numbers of asialyated receptors on the epithelial surface. Recent studies suggest that P. aeruginosa interacts with normal CFTR leading to ingestion by respiratory epithelial cells (Pier et al Science, 1996) which are then shed, possibly due to apoptosis. Both these processes may be relevant to the pathogenesis of infection in cystic fibrosis. We compared the trafficking of P. aeruginosa in CF and normal respiratory epithelial cells in primary culture and in transformed cell lines using flow cytometry, electron microscopy and confocal microscopy. To determine the relative contribution of superficial attachment versus epithelial phagocytosis, epithelial cell lines were grown in a polarized fashion on transwells, and treated with 1×108 cfu/ml of PAO1. When examined by EM polarized cells with tight junctions show minimal P. aeruginosa adherence and no invasion. Epithelial cells with disrupted tight junctions had increased intracellular bacterial invasion and evidence of intracellular replication. Flow cytometry was used to differentiate adherent from ingested bacteria, facilitating rapid analysis of a large number of individual cells. PAO1 labeled with green fluorescent protein (GFP) were incubated with monolayers of IB-3 (CF) and C-38 cells (“corrected”) cell lines. FACS analysis allowed the enumeration of all cells associated with bacteria and secondary labeling with Texas Red detected adherent but not ingested organisms. 18% of the CF cells had ingested organisms, which was half the amount ingested by the corrected cell line (44%). Ingestion of PAO1 was not abolished by cytochalasin-D. Confocal microscopy clearly differentiated green (ingested) from yellow (adherent) bacteria. These results contrasted markedly with the data obtained with primary cells which retained tight junctions and had virtually no internalized bacteria. A TUNEL assay demonstrated that epithelial cells with ingested bacteria were apoptotic within 6 hours. These experiments suggest P. aeruginosa can invade epithelial cells when the integrity of the epithelium is damaged but are only minimally adherent and non-invasive to an intact respiratory epithelium.