To investigate the epidemiology of nontypeable Haemophilus influenzae in the respiratory tract of cystic fibrosis (CF) patients, H. influenzae isolates from sputum specimens of 40 CF patients were analyzed longitudinally for 2 years. The isolates were characterized by analysis of the major outer membrane protein (MOMP) patterns. MOMP variant H. influenzae strains were discriminated from distinct strains by randomly amplified polymorphic DNA analysis of genomic DNA. Multiple H. influenzae strains and MOMP variant strains were isolated from single sputum specimens of 29 patients. In 22 patients, a distinct H. influenzae strain persisted over time (median persistence, 8 months; range 2-24). In general, the appearance of MOMP variant strains did not coincide with the occurrence of exacerbations.