A diagnostic assay has been developed to detect human cytomegalovirus (HCMV) DNA in clinical specimens with 32P-labeled cloned fragments of HCMV strain AD169. The labeled probe can detect 10 pg of HCMV DNA and fails to hybridize to DNA from other herpesviruses or human DNA. The assay correctly identified 22 (92%) of 24 coded urine specimens culture positive for HCMV and 23 (88%) of 26 urine specimens culture negative for HCMV. In a prospective study of 67 buffy-coat specimens from recipients of bone marrow transplants HCMV DNA was detected in 13 (93%) of 14 culture-positive samples. Of 53 buffy-coat specimens culture negative for HCMV, 32 were hybridization negative for HCMV DNA. However, in 20 of 21 buffy-coat specimens positive for HCMV by hybridization but negative by culture there was evidence that the hybridization assay was correct and more sensitive than currently available tissue culture techniques.