Although Hemophilus influenzae resistance to fluoroquinolones is low, it is important to monitor the development of populations with decreased susceptibility to this class of antimicrobials. The activity of gemifloxacin, a new fluoroquinolone, was measured against a collection of susceptible wild-type strains (MIC(90,) 0.008 microg/ml for both beta-lactamase-negative and -positive strains) and strains less susceptible to quinolones (MIC(90,) 1 microg/ml). Using a 5microg disk, the gemifloxacin-susceptible strains had MIC results ranging from </= 0.004 to 0.25 microg/ml (the proposed breakpoint), corresponding to zone diameters of 20 to 39 mm. However, some H. influenzae strains having multiple QRDR mutations were less susceptible to gemifloxacin (MIC range, 0.016-2 microg/ml), although gemifloxacin remained active against 57.1% of strains. In vitro testing, using these selected resistant strains, defined the categorical interpretive criteria for routine clinical laboratory use.