To determine whether 5% diphenhydramine solution has an anesthetic effect when administered topically to rabbit corneas.Experimental study.Twenty white New Zealand rabbits.Twenty rabbits at the University of Arkansas for Medical Sciences received 1 drop of 5% diphenhydramine solution in the left eye and 1 drop of balanced salt solution in the right eye. Corneal sensation was then measured with a Cochet-Bonnet esthesiometer at 30-, 60-, and 90-minute intervals. Rabbits were observed for conjunctival reaction. Follow-up fluorescein and Rose Bengal slit-lamp examinations were then performed to assess toxicity.Diphenhydramine solution at a 5% concentration demonstrated a significant anesthetic effect 30, 60, and 90 minutes after instillation (p < 0.0001, p = 0.0001, p = 0.0164, respectively). Mild conjunctival injection occurred in all diphenhydramine-treated eyes. No toxic effects on the corneal epithelium were observed.When applied topically to rabbit corneas, 5% diphenhydramine solution has a significant anesthetizing effect compared with salt solution (control eyes). Topical diphenhydramine may be a safe alternative in patients requiring topical anesthesia who have multiple allergies to topical anesthetics. Additional studies are needed to determine a dose-response curve and to further evaluate corneal toxicity prior to use in humans.