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Rose bengal and fluorescein are photosensitive dyes in widespread use in the evaluation of ocular surface diseases, including herpes simplex virus (HSV) keratitis. These dyes have recently been shown to penetrate living cells, and rose bengal was previously reported to possess antiviral activity. Several experiments reported herein suggest that these dyes do possess the potential for potent antiviral activity against extracellular virus, but only in the presence of light. Rose bengal is substantially more effective in vitro than fluorescein, and the effect is greater with increasing concentration of dye and duration of light exposure. Electron microscopic evaluation of treated virus showed no structural difference from untreated virus, in spite of 4- to 5-log decreases in virus titer. Intracellular virus was found to be markedly resistant to photoinactivation. In a rabbit model of acute primary HSV keratitis, daily application of topical rose bengal followed by light exposure had no therapeutic effect, although an adverse effect on culture sensitivity testing was seen.