A diagnostic hybridization assay for detecting herpes simplex virus type 1 (HSV-1) in ocular specimens was developed using cloned viral DNA as a probe. This hybridization assay is based on visualizing a biotinylated probe that is hybridized to the target DNA by a streptavidin/alkaline phosphatase system. The time required for performing this assay system is only two days. This assay system could detect a probe which had been hybridized to as little as 1 pg of homologous DNA and did not cross-react with DNA of other human herpes viruses except that of herpes simplex virus type 2 (HSV-2) which showed weak cross-reactivity. The assay system was applied to experimental keratitis in albino rabbits and clinical specimens. In experimental keratitis in rabbits it was possible to detect HSV-1 DNA in the eye swab samples at least until the ninth day after virus inoculation. Five clinical specimens collected from patients with corneal ulcer or blepharitis contained HSV-1 DNA in spite of the failure of demonstration of viral antigen and/or virus isolation in two cases.