The antibacterial activity of levofloxacin was compared with those of ofloxacin, ciprofloxacin, and other antibiotics. In general, levofloxacin was equally active or up to fourfold more active than ofloxacin against all 801 organisms tested. Levofloxacin was twofold [corrected] more active than ciprofloxacin against Streptococcus pneumoniae and 2- to 4-fold more active than ciprofloxacin against Staphylococcus aureus, Xanthomonas maltophilia, and Bacteroides fragilis. Levofloxacin was two- to eightfold more active than ciprofloxacin against coagulase-negative staphylococci and Acinetobacter spp., although these improvements in potency may not be clinically relevant. Levofloxacin inhibited 90% of streptococci when it was used at concentrations of 1 to 2 micrograms/ml. Levofloxacin was two- to fourfold less active than ciprofloxacin against most members of the family Enterobacteriaceae, such as Escherichia coli; Klebsiella pneumoniae; Citrobacter, Proteus, Providencia, Salmonella, and Yersinia spp.; and Pseudomonas aeruginosa. Both compounds were equally active against Pseudomonas cepacia. The in vitro DNA gyrase inhibitory activity of levofloxacin was as potent as that of ciprofloxacin, with a 50% inhibitory concentration of 0.65 micrograms/ml against an E. coli enzyme. In vivo, oral treatment with levofloxacin was as efficacious or more efficacious than that with ciprofloxacin in systemic as well as pyelonephritis infections in mice. Levofloxacin achieved higher concentrations in the serum and tissue of mice than did ciprofloxacin. This study presents some potential advantages of the pure L isomer of ofloxacin over ciprofloxacin and other quinolones.