β Interferon (lFN) was demonstrated by specific, sequential antibody-neutralization assays of vesicle fluids from patients with recurrent skin lesions due to herpes simplex virus. To determine the origin of this antiviral activity, we cultured keratinocytes from normal facial skin and infected them with three strains of herpes simplex virus. Keratinocyte cultures then developed characteristic cytopathic changes, and antiviral activity was found in culture supernatant media. All such activity from these supernatants was neutralized with specific antiserum to IFN-β but not with antiserum to IFN-α. No IFN-γ was detectable by radioimmunoassay. Immunoperoxidase staining with antiserum to IFN-β in five biopsy specimens from culture-proven, recurrent herpes simplex lesions showed positive staining of epidermal keratinocytes but not of dermal or infiltrating cells. Thus, the primary sources of IFN-β in recurrent herpes lesion vesicles are the virus-infected keratinocytes.