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Purpose: The objective of the present study was to evaluate the effectiveness of topically applied gemifloxacin for the treatment of experimental Staphylococcus aureus keratitis in a rabbit model., Methods: Rabbit corneas were intrastromally injected with ~100 colony-forming units (CFU) of S. aureus ATCC25923. Eight hours (early treatment) or 16 h (late treatment) after the injection, 1 topical drop of balanced salt solution (BSS), gemifloxacin ophthalmic solution (0.5%), levofloxacin ophthalmic solution (0.5%), or gatifloxacin eye gel (0.3%) was applied to each eye every 15 min for 5 doses and then, every 30 min for 14 doses. The eyes were examined both before and after treatment. The corneas were harvested from treated and untreated rabbits for the quantitation of bacteria and histological observation., Results: In the early-treatment groups, all 3 fluoroquinolones significantly lowered the clinical severity of infection and the median erosion area of the cornea compared with the BSS control (P=0.000). In the late-treatment groups, gemifloxacin and levofloxacin did not cause a significant reduction in clinical scores compared with the BSS control (P=0.107 and 0.531, respectively), but the gatifloxacin caused a significant reduction in clinical scores compared with the BSS control (P=0.011). The median erosion area significantly decreased with treatment with gemifloxacin, gatifloxacin, and levofloxacin in both early- and late-treatment groups, when compared with the control group (P≤0.022). In the early-treatment groups, the gemifloxacin, gatifloxacin, and levofloxacin groups had significantly lower CFU recovered from the corneas compared with the control group (P<0.01), while in the late-treatment groups, levofloxacin failed to reduce the CFU recovered from the corneas compared with the control group (P=0.695). The minimal inhibitory concentrations for gemifloxacin, gatifloxacin, and levofloxacin against S. aureus ATCC25923 were 0.0625, 0.0625, and 0.125 mg/L, respectively., Conclusions: Gemifloxacin, similar to gatifloxacin and levofloxacin, can significantly lower the clinical severity and CFU per cornea observed in S. aureus keratitis when early treatment is implemented. Significantly, gemifloxacin showed a significant efficacy improvement in reducing the bacterial load recovered from the corneas in the late-treatment experiment.