Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.