The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 10(7) copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.