Cells infected with HSV-1 or HSV-2 develop viral antigens which can be detected by immunofluorescence. We developed a flow cytometric indirect immunofluorescence assay to detect and quantitate antibodies to HSV-1 and HSV-2 in human sera. Results obtained by flow cytometry for detecting antibodies against HSV-1, when compared with results obtained by ELISA, showed an index of overall agreement of 100%. The correlation between the antibody titers obtained with each method was found to be highly significant. An index of overall agreement equal to 94.1% was observed between results obtained by flow cytometry and by immunofluorescence as concerns the discrimination of HSV-2 positive from negative samples. However, the correlation between antibody titers was found to be not statistically significant. The flow cytometric assay proved to be type-specific.