A rapid DNA-DNA hybridization technique that can be accomplished in 4 to 5 days was compared with plaque reduction assay to determine its reliability in performing antiviral assays for human cytomegalovirus (HCMV). The assay involves lysing infected cells, direct wicking of denatured DNA onto membranes and hybridization using a 125I-labeled HCMV DNA probe. Using ten ganciclovir sensitive clinical HCMV strains for comparison, the DNA hybridization technique correlated well with the plaque assay. Clinical HCMV strains previously identified as resistant to ganciclovir were also readily identified. The DNA-DNA hybridization assay is less tedious and more rapid than plaque reduction assays, and thus, provides an excellent alternative for evaluation of the antiviral activity of drugs against HCMV.