OBJECTIVE The use of topical fluoroquinolones to treat microbial keratitis is associated with an increased incidence of corneal perforation compared to other standard treatments. This study examined the effects of topical fluoro-quinolones on corneal collagen and keratocytes in intact rabbit corneas and corneas with an epithelial defect. METHODS Studies consisted of one group of intact corneas and one group of corneas where a 6-mm epithelial defect was created with a surgical scrape. Within each group, eyes were randomly assigned to one of four topical medications (0.3% ciprofloxacin, 0.3% ofloxacin, fortified antibiotics (1.36% tobramycin, 5% cefrazolin) or Tears Naturale (Alcon Laboratories, Frenchs Forest, NSW, Australia). Two drops were instilled hourly for 48 h and then 2-hourly for an additional 48 h. At 96 h the corneas were removed and processed for light microscopy, immunohistology for collagen IV, V and VI, and apoptosis staining. RESULTS In intact rabbit corneas there was no demonstrable difference between treatment groups. In corneas with an epithelial defect, both fluoroquinolones delayed epithelial healing when compared to fortified antibiotics or tears. Keratocyte loss was seen in all groups and was greatest in the ofloxacin group. Median stromal thickness with keratocyte loss were: ofloxacin 30%; ciprofloxacin 10%; fortified antibiotics 7.5%; and tears 15% (ofloxacin vs tears, Mann-Whitney = 16.0, P = 0.09). Keratocyte loss did not correlate with the amount of demonstrable apoptosis. Collagens IV, V and VI showed no differences between treatments. CONCLUSIONS These results suggest that ofloxacin is potentially cytotoxic to corneal keratocytes. Such an effect could lead to the observed increased incidence of corneal perforation in microbial keratitis.