Nontypeable Haemophilus influenzae colonizes the respiratory tract of adults with chronic obstructive pulmonary disease (COPD) and causes intermittent exacerbations. Isolates of H. influenzae collected monthly in a prospective study were subjected to molecular typing. During a 7-year study spanning 345 patient-months of observation, 122 episodes of negative cultures lasting 1 month or more, and that were preceded and followed by isolation of an apparently identical strain of H. influenzae, were found. Seventeen such episodes of negative cultures, lasting 6 months or more and spanning 203 patient-months, were studied in detail to test the hypothesis that these periods of negative cultures represented continuous colonization by the same strain of H. influenzae. Molecular typing by three independent methods established that the strains preceding and following the episodes of negative cultures were indeed identical. Strain-specific H. influenzae DNA was detected in some of the sputum samples that had yielded negative cultures. These results indicate that some patients with COPD are persistently colonized with H. influenzae and that sputum cultures underestimate the frequency of colonization of the respiratory tract by H. influenzae in COPD. This observation has a significant impact on understanding bacterial colonization in COPD.