We have previously reported the construction of a cell line BA4, constitutively producing the glycoproteins gD, gG, and alpha 4, the major regulatory protein of HSV-1. These cells have been selected in stepwise increasing concentrations of methotrexate and shown to produce much higher amounts of gD than non-selected cells. Extracts of the selected cells were used in an enzyme linked immunosorbent assay to detect HSV antibodies in human sera obtained from Greek blood donors. We report here that (i) the assay developed is able to distinguish HSV antibody positive from negative human sera and (ii) that its application in an epidemiological survey showed that the incidence of HSV infection in the general population in Greece is 90.4%.