The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of Haemophilus influenzae to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were >0.12 g/ml (range, 0.12 to 32 g/ml), and the ciprofloxacin MICs for 28 matched control isolates were <0.06 g/ml. In addition, we sequenced the quinolone resistance-determining regions (QRDRs) of gyrA and parC of all strains. The log 2 MICs of all quinolones were plotted against the inhibition zone diameters. The MICs and inhibition zone diameters selected to screen for the resistance mechanism were based on the susceptibility distribution data and the presence or absence of amino acid changes in the QRDRs of GyrA and ParC. Strains for which ciprofloxacin MICs were <0.06 g/ml, levofloxacin and moxifloxacin MICs were <0.03 g/ml, and nalidixic acid MICs were <2.0 g/ml lacked modifications in the QRDR of GyrA. In contrast, all strains for which ciprofloxacin, levofloxacin, and moxifloxacin MICs were >0.5 g/ml and the vast majority of those for which nalidixic acid MICs were >32 g/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen H. influenzae isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).Haemophilus influenzae is a common cause of pneumonia and other acute and chronic respiratory infections in children and adults. Although fluoroquinolones remain among the antimicrobial agents that are the most powerful against H. influenzae in vitro and are also highly effective as oral treatments for respiratory tract infections (15), resistance has been recognized (1,7,16,25). The emergence of Streptococcus pneumoniae and H. influenzae isolates with reduced susceptibilities to ciprofloxacin and other quinolones appears to be a growing problem worldwide for clinical and public health. Recently, therapeutic failures in patients with community-acquired pneumonia associated with levofloxacin resistance in S. pneumoniae (10) and H. influenzae (3) have been described.Cross-resistance to a large number of quinolones has been observed in H. influenzae (25), suggesting that testing for susceptibility to one quinolone would be sufficient to ascribe a loss of susceptibility to the entire antibiotic family in the majority of cases. On the basis of results of studies with a small number of isolates, we and others have suggested that subunit A of topoisomerase II (GyrA) and subunit A of topoisomerase IV (ParC) might be the first and second targets of quinolone action in H. influenzae, respectively (7,16,30).Nalidixic acid has received special attention for use in a scree...