We have performed a 4-year prospective surveillance for Pseudomonas aeruginosa cross-infection at a large regional adult cystic fibrosis center. Despite purpose-built facilities in a new building and the practice of strict hygiene, P. aeruginosa cross-infection has continued. In contrast, individuals segregated from the cohort of patients with chronic P. aeruginosa infection but who attend the same center have not acquired infection with transmissible P. aeruginosa strains. Simple infection control measures alone do not prevent the spread of transmissible P. aeruginosa strains between individuals with cystic fibrosis. However, in our clinic patient segregation effectively controlled spread of such strains.