Use of an antigen consisting of purified isolated nuclei from a mixture of human cytomegalovirus-infected and uninfected fibroblasts in a 2:1 ratio is a simple and reliable method for eliminating nonspecific fluorescence associated with the presence of Fc-immunoglobulin G receptors in the cytoplasm of infected cells. The specificity obtained with this antigen on 100 normal human sera was 99, 100, and 98% when compared with microneutralization, anticomplement immunofluorescence, and conventional indirect fluorescent-antibody assays, respectively. Also, 95% of the antibody titers obtained with the nuclear antigen had a perfect correlation with or were within a fourfold-dilution difference of the antibody levels obtained by anticomplement immunofluorescence and the conventional indirect fluorescent-antibody test.