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Purpose: To investigate the efficacy of the fourth-generation fluoroquinolone, gatifloxacin 0.3%, compared to ciprofloxacin 0.3%, in preventing Streptococcus pneumoniae keratitis in a rabbit laser in situ keratomileusis (LASIK) model., Methods: Twelve albino rabbits had bilateral lamellar flaps created. Group A (eight eyes) was given gatifloxacin 0.3%; group B (eight eyes) was given ciprofloxacin 0.3%; and group C (eight eyes) served as the controls. Groups A and B received one drop of antibiotic 20 minutes before the creation of the lamellar flap, at the conclusion of flap formation, and four times per day for 3 days. All corneas were inoculated with 0.1 mL of 4 x 10 organisms/mL of S. pneumoniae immediately after flap formation. On day 3, all corneas were examined and cultured., Results: Group A (gatifloxacin) had no infiltrates and three areas of 1-mm central corneal haze. On day 3, one of eight corneas had a positive culture. Group B (ciprofloxacin) had seven infiltrates, including one perforation, and six of eight corneas had positive cultures. Group C (control) had eight corneal infiltrates, and all eight corneas had positive cultures. The data show a statistically significant difference between gatifloxacin and ciprofloxacin and gatifloxacin and control for mean infiltrate size and mean culture scores., Conclusions: The fourth-generation fluoroquinolone, topical gatifloxacin 0.3%, is superior to topical ciprofloxacin 0.3% for prophylaxis against a clinical isolate of S. pneumoniae in a rabbit LASIK model.