To investigate the presence and the role of polyomaviruses JC (JCV), BK (BKV), and the simian polyomavirus (SV40) in human brain tumors, samples from 25 glial-derived tumors (10 astrocytomas, 5 ependymomas, 5 oligodendrogliomas, and 5 glioblastomas) were examined by means of molecular biology and immunohistochemistry. Nested PCR of the large T (LT) region and its sequence analysis showed JCV in 6 cases (4 astrocytomas, 1 oligodendroglioma, and 1 ependymoma), while the transcriptional control region (TCR) was amplified only in 1 astrocytoma, the oligodendroglioma, and the ependymoma, one of which (astrocytoma) also stained positively by immunohistochemistry (JCV LT). TCR sequence analysis of the oligodendroglioma showed a JCV rearranged structure not related to a known viral strain, while the astrocytoma and the ependymoma disclosed a JCV Mad-4 strain that is known to induce brain tumors in animals. We suggest that JCV could have played a role in the pathogenesis of these brain tumors.