Ocular penetration of two topical antibiotics used to treat bacterial conjunctivitis was assessed in adult volunteers scheduled for cataract surgery. In this randomized, parallel-group study, patients instilled trimethoprim sulfate 0.1%/polymyxin B (n = 23) or ofloxacin 0.3% (n = 25) QID for 3 days, plus 4 instillations in the hour before surgery. Analysis of aqueous humor samples obtained during surgery showed a 2.4-fold greater concentration of ofloxacin over trimethoprim (1.135 micro g/ml vs 0.470 micro g/ml; P <.0001). The greater concentration of ofloxacin in ocular tissue coupled with its superior antibacterial activity profile supports its use as an alternative to trimethoprim/polymyxin B for treatment of bacterial conjunctivitis.