Human brain tumors of 11 different types were analyzed by Southern blot analysis for the presence of JCV, SV40, and BKV. In 21 tumor specimens examined with JCV- and SV40-specific probes no positive hybridizations were obtained. Analysis for BKV DNA, however, revealed the presence of BKV-specific sequences in 11 of 24 tumor specimens. No hybridization was found in DNA from CNS tissues from different areas of 29 individuals without CNS tumors. The BKV DNA sequences were associated with high molecular weight cellular DNA, suggesting a chromosomal location. These data provide evidence for the involvement of BKV in the development of human brain tumors.