Credible but conflicting reports address the frequency of prenatal infection by species C adenovirus. This question is important because these viruses persist in lymphoid cells and suppress double-stranded DNA-break repair. Consequently, prenatal adenovirus infections may generate the aberrant clones of lymphocytes that precede development of childhood acute lymphoblastic leukemia (ALL). The present study was designed to overcome technical limitations of prior work by processing cord blood lymphocytes within a day of collection, and by analyzing sufficient numbers of lymphocytes to detect adenovirus-containing cells at the lower limits determined by our previous studies of tonsil lymphocytes. By this approach, adenoviral DNA was identified in 19 of 517 (3.7%) samples, providing definitive evidence for the occurrence of prenatal infection with species C adenoviruses in a significant fraction of neonates predominantly of African American and Hispanic ancestry. Cord blood samples were also tested for the presence of the ETV6-RUNX1 translocation, the most common genetic abnormality in childhood ALL. Using a nested PCR assay, the ETV6-RUNX1 transcript was detected in four of 196 adenovirus-negative samples and one of 14 adenovirus-positive cord blood samples. These findings indicate that this method will be suitable for determining concordance between adenovirus infection and the leukemia-associated translocations in newborns.