Staphylococcus aureus colonizes the lungs of cystic fibrosis patients and treatment with antibiotics usually results in recurrent and relapsing infections. We have shown that S. aureus can invade and replicate within a cystic fibrosis epithelial cell line (CFT-1), and that these internalized bacteria subsequently escape from the endocytic vesicle. The accessory gene regulator, agr, in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence. Here we show that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Trafficking analysis of live S. aureus which did not express alpha-haemolysin, a specific agr regulated toxin, revealed a defect in vesicular escape that was undistinguishable from the trafficking defect exhibited by the agr mutant. Furthermore, overexpression of alpha-haemolysin under an inducible promoter in an agr mutant of S. aureus partially restored the phagosome-escaping phenotype of an agr mutant. These results demonstrate that the expression of agr is required for vesicular escape, and that biologically active alpha-haemolysin is required for S. aureus escape from the endocytic vesicle into the cytosol of CFT-1 cells.