Herpes simplex virus (HSV) antigens in corneal or conjunctival epithelial lesions were detected by impression cytology. Specimens were obtained using a nitrocellulose membrane and were stained by the peroxidase-antiperoxidase method. HSV antigens were demonstrated in 30 of 32 patients with herpes simplex keratitis or conjunctivitis. Impression specimens of dendritic or stellate keratitis lesions exhibited precise replicas of corneal lesions with numerous antigen-positive cells. HSV antigens were also detectable in some of the minute stellate and/or punctate epithelial keratitis lesions even during the course of antiviral treatment. Impression cytology is noninvasive and useful for a rapid etiological diagnosis of herpetic epithelial lesions.