Adenoviruses (AdVs) contain genes coding for proteins with transforming potential, and certain AdV serotypes have been shown to induce tumors in rodents. However, data on the possible oncogenicity of AdVs in humans are scarce. We have therefore employed a real-time quantitative PCR (RQ-PCR) assay permitting highly sensitive detection of all 51 currently known human AdV serotypes to screen more than 500 tumor specimens derived from 17 different childhood cancer entities including leukemias, lymphomas, and solid tumors. Most tumor entities analyzed showed no evidence for the presence of AdV sequences, but AdV DNA was detected by RQ-PCR in different brain tumors including 25/30 glioblastomas, 22/30 oligodendrogliomas, and 20/30 ependymomas. Nonmalignant counterparts of AdV-positive brain tumors, including specimens of ependymal cells, plexus choroideus, and periventricular white matter, were screened for control purposes and revealed the presence of AdV DNA in most specimens tested. Identification of the AdV types present in positive malignant and nonmalignant brain tissue specimens revealed predominantly representatives of species B and D and, less commonly, C. To exclude contamination as a possible cause of false-positive results, specimens with AdV sequences detectable by PCR were subsequently analyzed by in situ hybridization, which confirmed the PCR findings in all instances. The central nervous system appears to represent a common site of AdV infection with virus persistence, thus providing the first evidence for the possible contribution of AdVs to the multistep process of tumor pathogenesis in brain tissue.