Pseudomonas aeruginosa lung infection is an important cause of morbidity and mortality in cystic fibrosis (CF). Longitudinal assessment of the phenotypic changes in P. aeruginosa isolated from young children with CF is lacking. This study investigated genotypic and phenotypic changes in P. aeruginosa from oropharynx (OP) and bronchoalveolar lavage fluid (BALF) in a cohort of 40 CF patients during the first 3 years of life; antibody response was also examined. A high degree of genotypic variability was identified, and each patient had unique genotypes. Early isolates had a phenotype distinct from those of usual CF isolates: generally nonmucoid and antibiotic susceptible. Genotype and phenotype correlated between OP and BALF isolates. As determined by culture, 72.5% of patients demonstrated P. aeruginosa during their first 3 years. On the basis of combined culture and serologic results, 97.5% of patients had evidence of infection by age 3 years, which suggests that P. aeruginosa infection occurs early in CF and may be intermittent or undetectable by culture.