The authors have developed an objective method for quantitation of herpes simplex virus in the corneal epithelium of rabbits. At appropriate times postinfection, full-thickness rabbit corneas were removed by trephination and subjected to one cycle of freezing and thawing. The corneal epithelium was then disrupted by sonication. The amount of infectious virus recovered from sonicated specimens was determined by an in vitro plaque assay, providing a measure of the quantity of virus present during the acute stage of herpetic keratitis. Using this technique, the authors found that the mean virus titer was reduced from 1.5 X 10(5) plaque forming units (pfu) per cornea in control rabbits to less than 200 pfu per cornea in rabbits treated topically for 2 days with 1% trifluridine. In contrast, instillation of 1% prednisolone acetate resulted in the persistence of higher levels of virus (275 pfu) than those observed in control rabbits (3 pfu) 4 days after the cessation of therapy.