The use of conventional methods to detect a possible infectious cause of Kawasaki disease (KD) has been unsuccessful. Using the polymerase chain reaction and DNA hybridization techniques, we have sought evidence that a known or new herpesvirus, parvovirus, or bacterial pathogen is related etiologically to KD. Peripheral blood DNA from acute KD patients was subjected to amplification and dot-blot hybridization to detect the presence of herpesvirus DNA, and acute KD peripheral blood and serum DNA were subjected to dot-blot hybridization for the presence of parvoviral DNA. All samples were negative for both herpesvirus and parvovirus DNA. In addition, we analyzed buffy-coat white blood cell DNA, synovial fluid DNA, and frozen autopsy and formalin-fixed, paraffin-embedded myocardial tissue DNA from KD patients for the presence of highly conserved bacterial 16S ribosomal RNA gene sequences with the polymerase chain reaction, and all were negative. These results argue against a direct pathogenic role for herpesviruses, parvoviruses, and bacteria in KD. This approach to the detection of highly conserved genomic sequences among broad groups of microorganisms can be adapted for the detection of other groups of microorganisms and may yet prove useful in the search for an etiologic agent of KD.