Ciprofloxacin has been a major advance in the treatment of chronic respiratory infections. Three patients with cystic fibrosis and colonized by 5 nontypeable Haemophilus influenzae strains exhibiting low- (MIC, 2 microg/mL) and high-level ciprofloxacin resistance (MICs, 16-32 microg/mL) are described. The patients had received several courses of ciprofloxacin. These MICs represent a decrease in ciprofloxacin susceptibility of 200-3200 times. Molecular epidemiologic methods demonstrated that 2 patients were chronically colonized by their own ciprofloxacin-resistant strains for > or = 15-17 months. Three strains showed simultaneous resistance to ampicillin and chloramphenicol by enzyme inactivation, and 2 had ampicillin resistance without beta-lactamase activity. These data suggest that the emergence and long-term persistence of ciprofloxacin-resistant H. influenzae in patients with cystic fibrosis can be a consequence of antibiotic treatment.