We assessed the nature and frequency of genome alterations in Staphylococcus aureus during chronic lung infection in patients with cystic fibrosis (CF) and during colonization of the nares in healthy individuals. Only individuals harboring the same S. aureus clone on consecutive samplings were included in the present study. Clone definition was based on pulsed-field gel electrophoresis (PFGE) analysis. Minor fragment variations in consecutive clones were interpreted as genome alterations. The frequency of genome alterations was significantly higher in S. aureus derived from patients with CF (mean time, 1.03 years) than in isolates derived from healthy individuals (mean time, 13.4 years). In total, 19 S. aureus strain pairs showing genome alterations were available for molecular analysis to clarify the nature of recombinational events in the host environment. In 8 cases, genome alteration could be linked to phage mobilization. Phage conversion of beta-toxin production was evident in 7 pairs. In 1 strain pair, changes in the PFGE pattern were accompanied by deletion of a phage similar to ETA. Obviously, phage mobilization plays an important role in vivo. During long-term lung infection in patients with CF, the specific host response and/or the regular exposure to antibiotics exercises strong selective pressure on the pathogen. Genome plasticity may facilitate the adaptation to various host conditions.