A polymerase chain reaction (PCR) assay was developed and optimized to detect cytomegalovirus (CMV) DNA in the blood of 86 normal donors who had originally tested seropositive for CMV. Evidence of previous or current infection with CMV was determined by rescreening of the blood for CMV antibodies and by detecting the presence of infectious virus in the white cells by cell culture. DNA was extracted from the blood of donors by a manual or an automated method and amplified by PCR using primers from the major immediate early gene of CMV DNA. The amplified product was detected by visualization of a fluorescent 435-base pair DNA band in an electrophoretic agarose gel after ethidium bromide staining and confirmed by slot-blot DNA hybridization using an oligonucleotide probe with complementarity for the major immediate early gene. Seven (8%) of the 86 donors were positive for CMV DNA in both fluorescence and hybridization studies. These donors were also antibody positive. While 74 (86%) of the 86 donors were positive for the presence of CMV antibodies in enzyme-linked immunosorbent assay, none was positive for virus in cell culture. PCR has the potential to be an effective and reliable procedure for the detection of CMV DNA in donor blood, but further study is required for this technique to be used for diagnostic or routine screening purposes.