The development of selective inhibitors of human cytomegalovirus (CMV) should lead to a better understanding of the mode of replication of CMV. A flow cytometric method was developed to monitor the expression of CMV antigens in CMV-infected human embryonic lung fibroblasts. The procedure is based on an indirect immunofluorescence assay using monoclonal antibodies directed against CMV-specific (immediate early, early, late) antigens and goat anti-murine IgG labeled with fluorescein isothiocyanate. Flow cytometric analysis clearly distinguished between the uninfected and infected cell population. There was a time-dependent appearance of CMV-specific antigens and a close correlation between the multiplicity of infection and the ratio of infected to uninfected cells. The method allows an accurate determination of the percentage of CMV-infected cells in the whole cell population and of the time of appearance of the viral antigens.