The in vitro activity of difloxacin (A-56619) and A-56620, two new aryl-difluoroquinolones, was compared to that of other new quinolones and several parenteral and oral antimicrobial agents. A-56620 inhibited 90% of Enterobacteriaceae at ≤1 μg/ml, Staphylococcus aureus 0.25 μg/ml, hemolytic streptococci 2 μg/ml, Pseudomonas aeruginosa 2 μg/ml, Bacteroides sp. and Clostridium at 8 μg/ml.A-56620 was equal or 2-fold more active than norfloxacin and ofloxacin, and 2–8-fold less active than ciprofloxacin. Difloxacin had similar in vitro activity with many isolates but usually was 2–8-fold less active than A-56620. Both agents inhibited β-lactamase positive Haemophilus influenzae (MIC 0.015 μg/ml)and Neisseria gonorrhoeae (MK ≤0.008 μg/ml).Both agents were more active against streptococci and Streptococcus pneumoniae than norfloxacin, ofloxacin and enoxacin, but not more active than ciprofloxacin. They inhibited Enterobacter cloacae, Citrobacter freundii and Serratia marcescens resistant to cephalosporins and methicillin-resistant S. aureus and Staphylococcus epidermidis. Spontaneously resistant mutants were seen with Enterobacteriaceae, P. aeruginosa and S. aureus at a frequency similar to that found for other new quinolones. These agents show overall in vitro activity comparable to other quinolones in clinical trial or recently approved for clinical use.