This study investigates the hypothesis that Hirschsprung's disease (HD), congenital rectal aganglionosis, may be etiologically linked to antenatal cytomegalovirus (CMV) infection. Bowel specimens from 72 HD patients, 144 control I infants (Hirschsprung-like symptoms, normal histology), and 36 control II infants (deaths from nongastrointestinal causes) were analyzed for CMV genomes by polymerase chain reaction. Positive results were obtained in 6 HD patients (8.8%) and none of the controls. Our findings suggest that antenatal CMV infection, a potentially preventable condition, may be one of the etiological factors in HD.