Detection of human cytomegalovirus (CMV) DNA from peripheral blood and urine specimens taken from renal transplant and bone marrow transplant recipients was examined by using nested polymerase chain reaction (PCR). Results by the PCR were compared with those of healthy subjects. Oligonucleotide primers were used to amplify the immediate-early (IE) and the late antigen (V) genes of CMV. Amplified DNA products were identified by 2% agarose gel electrophoresis and by Southern hybridization with alkaline phosphatase-labeled probes. Twenty eight blood specimens from 118 healthy subjects were positive for the V gene fragment of CMV, whereas the IE gene fragment was more associated with a negative result (116 of 118 specimens). When amplified with mixed primer pairs, no blood and urine specimens simultaneously produced the PCR products of two separate CMV genes. In the case of transplant patients, however, 50 of 139 blood specimens and 34 of 117 urine specimens were positive with the V primers, and 23 blood and 17 urine specimens were positive with the IE primers. Using mixed primer pairs, amplification of two CMV genes was detected in 14 blood and 10 urine specimens. In three cases of bone marrow transplant recipients, amplification of two CMV genes was detectable prior to anti-CMV IgM development. From these results, we suggest that detection of CMV-DNA by nested PCR with mixed primer pairs may be a valuable tool for diagnosing CMV infections.