In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC₉₀], 0.03 µg/ml; 100.0% of strains inhibited at 2 µg/ml) was the most active agent tested against H. influenzae and was similar to four comparison fluoroquinolones (MICs, 0.03 to 2 µg/ml) against M. catarrhalis. A subset of 300 recent clinical isolates of H. influenzae were tested by using media (Haemophilus Test Medium agar and broth) and procedures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) and with the E-test (AB BIODISK, Solna, Sweden). Gatifloxacin (MIC₅₀, 0.008 µg/ml) was slightly more active than levofloxacin, and E-test results were generally elevated by 0.5 log₂ dilution step compared to reference MICs. The gatifloxacin 5-µg disk test produced zone diameters that were routinely above 30 mm for H. influenzae strains, corresponding to gatifloxacin MICs of 0.008 or 0.016 µg/ml. The gatifloxacin susceptibility breakpoint proposed for nonfastidious species (2 µg/ml; 18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 µg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).