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Chlorhexidine gluconate is a bisguanide germicide currently available with 70% isopropanol (Hibistat, Hibitane) or a detergent (Hibiclens, Hibiscrub) for preoperative skin preparation. As these solvents are toxic to the cornea, we investigated the safety and efficacy of aqueous chlorhexidine solutions for ophthalmic use. Chlorhexidine in Tris-glycine buffer was evaluated for retardation of epithelial regeneration after experimental corneal abrasion in rabbits. Irrigant concentrations of 2.0 and 4.0% chlorhexidine significantly slowed the healing rate (0.546 and 0.076 mm/h, respectively) compared with saline controls (0.938 mm/h). Irrigant concentrations of less than or equal to 1% did not statistically delay healing (P greater than 0.4). In a separate group of animals, the right conjunctivae of pigmented rabbits were inoculated with Staphylococcus epidermidis (approximately 10(7) organisms per eye) and irrigated with 40 microliters of aqueous chlorhexidine in concentrations of 0.1, 0.5, and 1.0%; the left eyes were irrigated with saline or left untreated. Quantitative conjunctival cultures were obtained, and the total number of organisms recovered per eye was calculated. All chlorhexidine-treated eyes showed significant reduction in organisms compared with either untreated or saline-irrigated control eyes (P less than 0.001). In vitro antimicrobial susceptibility testing demonstrated chlorhexidine in concentrations of 0.1 to 4% to be highly active against a variety of gram-positive and gram-negative bacterial pathogens by disk diffusion and broth diffusion assays. Topical aqueous chlorhexidine may be an alternate agent for preoperative conjunctival antisepsis.