ABSTRACTStaphylococcus aureusis frequently the initial bacterium isolated from young cystic fibrosis (CF) patients, and yet its role in CF disease progression has not been determined. Recent data from our lab demonstrates thatS. aureuscan invade and replicate within the CF tracheal epithelial cell line (CFT-1). Here we describe the finding that the fate of internalizedS. aureusin CFT-1 cells differs from its complemented counterpart (LCFSN).S. aureusstrain RN6390 was able to replicate within the mutant CFT-1 cells after invasion but not in the complemented LCFSN cells. At 1 h postinvasion,S. aureuscontaining vesicles within both cell lines acquired vacuolar-ATPase, lysosomal markers LAMP 1 and 2, and the lysomotrophic dye LysoTracker to a similar degree. However, at 4 h postinvasion, the percentage ofS. aureuswithin CFT-1 cells associated with these markers decreased significantly compared to LCFSN, where the association approached 100%. Transmission electron microscopic analysis revealed that the majority of bacteria within CFT-1 cells were free in the cytosol at 4 h after invasion, whereas mostS. aureusbacteria internalized by LCFSN cells remained within vesicles. These results demonstrate a fundamental difference in the fate of liveS. aureusafter invasion of CFT-1 versus LCFSN cell lines and may explain the propensity ofS. aureusto cause chronic lung infection in CF patients.