An in vitro DNA hybridization assay was used to test 281 newborns for congenital infection with cytomegalovirus. The assay utilized an abbreviated method for DNA preparation and a dot blot assay that provided good sensitivity (100%) and specificity (98.9%) when compared with standard tissue culture, yet substantially reduced the total time of analysis. This assay would be a useful adjunct to tissue culture to diagnose newborns with congenital infection with cytomegalovirus.