We hypothesized that in adults with cystic fibrosis, the acquisition of a new strain of Pseudomonas aeruginosa may be associated with a pulmonary exacerbation. Eighty-four patients who were chronically infected with P. aeruginosa were prospectively followed from eight centers over a 26-month period. Patients had sputum cultures performed every 3 months while clinically stable and at the time of an exacerbation. Forty patients (48%) had an exacerbation requiring intravenous antibiotics during the study period, and in 36 of these patients, their P. aeruginosa isolates were genetically typeable by pulsed-field gel electrophoresis. In 34 of the 36 patients (94%), P. aeruginosa recovered during clinical stability and at exacerbation were of the same genotype. In only two patients (6%; 95% confidence interval, 0–18%) was a new P. aeruginosa clone cultured during an exacerbation that had not been cultured during clinical stability. There were no significant differences in antibiotic susceptibilities, measured as mean minimal inhibitory concentrations, for isolates retrieved during clinically stable periods compared with isolates retrieved during exacerbations. We conclude that for the majority of adult patients with cystic fibrosis a new pulmonary exacerbation is not caused by the acquisition of a new strain of P. aeruginosa.